Engineered microcompartment protein and related methods and systems of engineering bacterial systems for non-native protein expression and purification

ABSTRACT

Engineered microcompartment proteins, and related engineered microcompartment, vectors, cells compositions, methods and systems configured to provide within a cell one or more proteins non-native to the cell are described, wherein the one or more proteins non-native to the cell are contained in at least one engineered microcompartment within the cell.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional application No. 62/598,984, entitled “Engineered Microcompartment Protein and Related Methods and Systems of Engineering Bacterial Systems for Non-native Protein Expression and Purification” filed on Dec. 14, 2017, with docket number IL-13262, which is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT GRANT

The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC, for the operation of Lawrence Livermore National Laboratory.

FIELD

The present disclosure relates to protein production in cell systems and in particular to engineering microcompartment proteins and related bacterial systems for non-native protein expression and purification and related cells, compositions, methods and systems.

BACKGROUND

Production of non-native proteins has been the subject of research in several fields, including commercial and academic fields in connection with applications involving production in a cell of a protein non-native to that cell.

Despite the presence of various approaches, expression, production and/or purification in a cell of proteins non-native to said cell for various uses is still challenging.

SUMMARY

Provided herein, are engineered microcompartment proteins and related engineered microcompartments, vectors, cells, compositions, methods and systems that can be used in several embodiments for non-native protein expression, production and/or purification.

According to a first aspect, an engineered microcompartment protein is described. The engineered microcompartment protein comprises an encapsulin protein having sequence

-   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇—X₁₈—X₁₈—X₂₀—X₂₁—X₂₂—X₂₃—X₂₄—X₂₅—X₂₆—X₂₇—X₂₈—X₂₉—X₃₀     X₃₁—X₃₂—X₃₃     X₃₄—X₃₅—X₃₆—X₃₇—X₃₈—X₃₉—X₄₀—X₄₁—X₄₂—X₄₃—X₄₄—X₄₅—X₄₆—X₄₇—X₄₈—X₄₉—X₅₀—X₅₁—X₅₂—X₅₃—X₅₄—X₅₅—X₅₆—X₅₇—X₅₈—X₅₉—X₆₀—X₆₁—X₆₂—X₆₃—X₆₄—X₆₅—X₆₆—X₆₇—X₆₈—X₆₉—X₇₀—X₇₁—X₇₂—X₇₃—X₇₄—X₇₅—X₇₆—X₇₇—X₇₈—X₇₉—X₈₀—X₈₁—X₈₂—X₈₃—X₈₄X₈₅—X₈₆—X₈₈—X₈₉—X₉₀—X₉₁—X₉₂—X₉₃—X₉₄—X₉₅—X₉₆—X₉₇—X₉₈—X₉₉—X₁₀₀—X₁₀₁—X₁₀₂—X₁₀₃—X₁₀₄—X₁₀₅—X₁₀₆—X₁₀₇—X₁₀₈—X₁₀₉—X₁₁₀—X₁₁₁—X₁₁₂—X₁₁₃—X₁₁₄—X₁₁₅—X₁₁₆—X₁₁₇—X₁₁₈—X₁₁₉—X₁₂₀—X₁₂₁—X₁₂₂X₁₂₃—X₁₂₄—X₁₂₅—X₁₂₆—X₁₂₇—X₁₂₈—X₁₂₉—X₁₃₀—X₁₃₁—X₁₃₂—X₁₃₃—X₁₃₄—X₁₃₅—X₁₃₆—X₁₃₇—X₁₃₈—X₁₃₉—X₁₄₀—X₁₄₁—X₁₄₂—X₁₄₃—X₁₄₄—X₁₄₅—X₁₄₆—X₁₄₇—X₁₄₈—X₁₄₉—X₁₅₀—X₁₅₁—X₁₅₂—X₁₅₃—X₁₅₄—X₁₅₅—X₁₅₆—X₁₅₇—X₁₅₈—X₁₅₉—X₁₆₀—X₁₆₁—X₁₆₂—X₁₆₃—X₁₆₄—X₁₆₅—X₁₆₆—X₁₆₇—X₁₆₈—X₁₆₉—X₁₇₀—X₁₇₁—X₁₇₂—X₁₇₃—X₁₇₄—X₁₇₅—X₁₇₆—X₁₇₇—X₁₇₈—X₁₇₉—X₁₈₀—X₁₈₁—X₁₈₂—X₁₈₃—X₁₈₄—X₁₈₅—X₁₈₆—X₁₈₇—X₁₈₈—X₁₈₉—X₁₉₀—X₁₉₁—X₁₉₂—X₁₉₃—X₁₉₄—X₁₉₅—X₁₉₆—X₁₉₇—X₁₉₈—X₁₉₉—X₂₀₀—X₂₀₁—X₂₀₂—X₂₀₃—X₂₀₄—X₂₀₅—X₂₀₆—X₂₀₇—X₂₀₈—X₂₀₉—X₂₁₀—X₂₁₁—X₂₁₂—X₂₁₃—X₂₁₄—X₂₁₅—X₂₁₆—X₂₁₇—X₂₁₈—X₂₁₉—X₂₂₀—X₂₂₁—X₂₂₂—X₂₂₃—X₂₂₄—X₂₂₅—X₂₂₆—X₂₂₇—X₂₂₈—X₂₂₉—X₂₃₀—X₂₃₁—X₂₃₂—X₂₃₃—X₂₃₄—X₂₃₅—X₂₃₆—X₂₃₇—X₂₃₈—X₂₃₉—X₂₄₀—X₂₄₁—X₂₄₂—X₂₄₃     —X₂₄₄—X₂₄₅—X₂₄₆—X₂₄₇—X₂₄₈—X₂₄₉ X₂₅₀—X₂₅₁—X₂₅₂—X₂₅₃ (SEQ ID NO: 1) in     which X₁ is M, X₂ is D, X₃ is N, X₄ is L, X₅ is K, X₆ is R, X₇ is E,     X₈ is L, X₉ is A, X₁₀ is P, X₁₁ is L, X₁₂ is T, X₁₃ is E, X₁₄ is E,     X₁₅ is A, X₁₆ is W, X₁₇ is A, X₁₈ is E, X₁₉ is I, X₂₀ is D, X₂₁ is     E, X₂₂ is E, X₂₃ is A, X₂₄ is R, X₂₅ is E, X₂₆ is T, X₂₇ is A, X₂₈     is K, X₂₉ is R, X₃₀ is H, X₃₁ is L, X₃₂ is A, X₃₃ is G, X₃₄ is R,     X₃₅ is R, X₃₆ 1S V, X₃₇ 1S V, X₃₈ is D, X₃₉ 1S V, X₄₀ is E, X₄₁ is     G, X₄₂ is P, X₄₃ is L, X₄₄ is G, X₄₅ 1S W, X₄₆ is G, X₄₇ is Y, X₄₈     is S, X₄₉ is A, X₅₀ 1S V, X₅₁ is P, X₅₂ is L, X₅₃ is G, X₅₄ is R,     X₅₅ is L, X₅₆ is E, X₅₇ is E, X₅₈ is I, X₅₉ is E, X₆₀ is G, X₆₁ is     P, X₆₂ is A, X₆₃ is E, X₆₄ is G, X₆₅ is V, X₆₆ is Q, X₆₇ is A, X₆₈     is G, X₆₉ is V, X₇₀ is R, X₇₁ is Q, X₇₂ is V, X₇₃ is L, X₇₄ is P,     X₇₅ is L, X₇₆ is P, X₇₇ is E, X₇₈ is L, X₇₉ is R, X₈₀ is V, X₈₁ is     P, X₈₂ is F, X₈₃ is T, X₈₄ is L, X₈₅ is 5, X₈₆ is R, X₈₇ is R, X₈₈     is D, X₈₉ is L, X₉₀ is D, X₉₁ is A, X₉₂ is V, X₉₃ is E, X₉₄ is R,     X₉₅ is G, X₉₆ is A, X₉₇ is K, X₉₈ is D, X₉₉ is L, X₁₀₀ is D, X₁₀₁ is     L, X₁₀₂ is S, X₁₀₃ is P, X₁₀₄ is V, X₁₀₅ is VA, X₁₀₆ is E, X₁₀₇ is     A, X₁₀₈ is A, X₁₀₉ is R, X₁₁₀ is L, X₁₁₁ is L, X₁₁₂ is A, X₁₁₃ is R,     X₁₁₄ is A, X₁₁₅ is E, X₁₁₆ is D, X₁₁₇ is R, X₁₁₈ is L, X₁₁₉ is I,     X₁₂₀ is F, X₁₂₁ is N, X₁₂₂ is G, X₁₂₃ is Y, X₁₂₄ is A, X₁₂₅ is E,     X₁₂₆ is A, X₁₂₇ is G, X₁₂₈ is I, X₁₂₉ is E, X₁₃₀ is G, X₁₃₁ is L,     X₁₃₂ is L, X₁₃₃ is N, X₁₃₄ is A, X₁₃₅ is S, X₁₃₆ is G, X₁₃₇ is N,     X₁₃₈ is L, X₁₃₉ is K, X₁₄₀ is L, X₁₄₁ is P, X₁₄₂ is L, X₁₄₃ is S,     X₁₄₄ is A, X₁₄₅ is D, X₁₄₆ is P, X₁₄₇ is G, 3 X₁₄₈ is D, X₁₄₉ is I,     X₁₅₀ is P, X₁₅₁ is D, X₁₅₂ is A, X₁₅₃ is I, X₁₅₄ is A, X₁₅₅ is E,     X₁₅₆ is A, X₁₅₇ is L, X₁₅₈ is T, X₁₅₉ is K, X₁₆₀ is L, X₁₆₁ is R,     X₁₆₂ is E, X₁₆₃ is A, X₁₆₄ is G, X₁₆₅ is V, X₁₆₆ is E, X₁₆₇ is G,     X₁₆₈ is P, X₁₆₉ is Y, X₁₇₀ is A, X₁₇₁ is L, X₁₇₂ is V, X₁₇₃ is L,     X₁₇₄ is S, X₁₇₅ is P, X₁₇₆ is D, X₁₇₇ is L, X₁₇₈ is Y, X₁₇₉ is T,     X₁₈₀ is A, X₁₈₁ is L, X₁₈₂ is F, X₁₈₃ is R, X₁₈₄ is V, X₁₈₅ is Y,     X₁₈₆ is D, X₁₈₇ is G, X₁₈₈ is T, X₁₈₉is G, X₁₉₀ is Y, X₁₉₁ is P,     X₁₉₂ is E, X₁₉₃ is I, X₁₉₄ is E, X₁₉₅ is H, X₁₉₆ is I, X₁₉₇ is K,     X₁₉₈ is E, X₁₉₉ is L, X₂₀₀ is V, X₂₀₁ is D, X₂₀₂ is G, X₂₀₃ is G,     X₂₀₄ is V, X₂₀₅ is I, X₂₀₆ is W, X₂₀₇ is A, X₂₀₈ is P, X₂₀₉ is A,     X₂₁₀ is L, X₂₁₁ is D, X₂₁₂ is G, X₂₁₃ is G, X₂₁₄ is A, X₂₁₅ is V,     X₂₁₆ is L, X₂₁₇ is V, X₂₁₈ is S, X₂₁₉ is T, X₂₂₀ is R, X₂₂₁ is G,     X₂₂₂ is G, X₂₂₃ is D, X₂₂₄ is F, X₂₂₅ is D, X₂₂₆ is L, X₂₂₇ is T,     X₂₂₈ is L, X₂₂₉ is G, X₂₃₀ is Q, X₂₃₁ is D, X₂₃₂ is L, X₂₃₃ is S,     X₂₃₄ is I, X₂₃₅ is G, X₂₃₆ is Y, X₂₃₇ is L, X₂₃₈ is S, X₂₃₉ is H,     X₂₄₀ is D, X₂₄₁ is A, X₂₄₂ is D, X₂₄₃ is N, X₂₄₄ is V, X₂₄₅ is E,     X₂₄₆ is L, X₂₄₇ is F, X₂₄₈ is L, X₂₄₉ is T, X₂₅₀ is E, X₂₅₁ is S,     X₂₅₂ is F, X₂₅₃ is T (SEQ ID NO: 1) -   or a sequence with at least 22% sequence identity or at least 40%     sequence similarity to SEQ ID NO: 1.

In the engineered microcompartment protein, the encapsulin protein comprises a P-Domain (Peripheral Domain), an E-Loop (Elongated Loop) and an A-Domain (Axial Domain).

In the engineered microcompartment protein, the P-Domain of the encapsulin protein comprises

a first fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 26 residues alpha helix αl, linked to a 0 to 22 residues first non-structured region (such as a loop region), linked to a 4 to 11 residues alpha helix α2, linked to a 3 to 9 residues beta strand β1, linked to a 3 to 13 residues second non-structured region.

a second fragment of the P-Domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising a direction N-terminus to C-terminus a 9 to 15 residues beta strand (34, linked to a 6 to 15 residues alpha helix α3, linked to a 0 to 10 residues first non-structured region, a 18 to 29 residues alpha helix α4, and a 9 to 21 residues second non-structured region; and

a third fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded microcompartment protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 10 residues beta strand β9, linked to a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand β10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand β11.

In the engineered microcompartment protein, the E-Loop of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 8 to 16 residues beta strand β2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand β3, linked to a 0 to 6 residues second non-structured region.

In the engineered microcompartment protein, the A-Domain of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 0 to 8 residues beta strand β5 linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix α5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand β6, linked to a 9 to 16 residues alpha helix α6, linked to a 1 to 24 third non-structured region, linked to a 0 to 16 residues alpha helix α7, linked to a 0 to 8 residues fourth non-structured region, linked to a 1 to 10 residues beta strand β7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand β8, linked to a 2 to 12 residues sixth non-structured region.

In the engineered microcompartment protein, the P-domain, A-domain and E-loop are arranged together in a configuration comprising, in a direction N-terminus to C-terminus, the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain.

In the engineered microcompartment protein,

a target protein having an N-terminus, a C-terminus is inserted at the N-terminus of the first segment of the P-domain of the encapsulin protein alone or together with a linker and/or a tag

at least one first protease cleavage site is inserted between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein alone or together with a linker and/or a tag; and

at least one second protease cleavage site is inserted at the C-terminus of the E-loop of the encapsulin protein or within 1-17 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein, alone or together with a linker and/or a tag, to enable digestion of the encapsulin and release of the target protein.

In some embodiments wherein the engineered microcompartment proteins are designed to be non cage forming proteins, the at least one second protease cleavage site is inserted at the C-terminus of the E-loop of the encapsulin protein or within 1-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein (within β3 of the E-loop).

In some embodiments wherein the engineered microcompartment proteins are designed for cage forming proteins, the at least one second protease cleavage site can be inserted within 9-17 amino acids from the C-terminus of the E-loop of the encapsulin protein (within the loop region between β2 and β3 of the E-loop), and/or inserted within 2-14 amino acids adjacent to the N-terminus of the A-domain (in the flexible region between the N-terminus of the A-domain and α5, including β5).

In some embodiments, the target protein is a protein non-native to one or more bacterial cell (herein also indicated as non-native protein) and capable of causing cell damage (herein also indicated as non-native toxic protein). In some embodiments the at least one first protease and the at least one second protease cleavage site can be same or different.

According to a second aspect, an engineered microcompartment is described, the engineered microcompartment comprising a same or different at least one engineered microcompartment protein herein described. In particular, in some embodiments, the engineered microcompartment proteins of the engineered microcompartment have a same target protein, at least one first protease cleavage site and/or at least one second protease cleavage site.

According to a third aspect, a method is described to produce in a bacterial cell a protein non-native to the bacterial cell. The method comprises introducing into the bacterial cell at least one first polynucleotide encoding at least one engineered microcompartment protein herein described in which the target protein is the protein non-native to the bacterial cell. In the method, the at least one first polynucleotide is operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the bacterial cell. In the method the introducing is performed to obtain expression in the bacterial cell of the at least one engineered microcompartment protein to obtain the protein non-native to the bacterial within at least one engineered microcompartment formed by the at least one engineered microcompartment protein.

In some embodiments, the protein non-native to the bacterial cell is a toxic non-native protein capable of reacting with a native membrane substrate of the bacterial cell with a reaction resulting in a damage of the bacterial cell, and the engineered microcompartment protein is provided in the bacterial cell to shield the bacterial cell from toxicity during intracellular production of the toxic non-native protein in the bacterial cell.

According to a fourth aspect, a system is described to produce, in bacterial cell, a protein non-native to the bacterial cell. The system comprises

at least one first polynucleotide encoding at least one engineered microcompartment protein herein described wherein the target protein is the protein non-native to the bacterial cell, the at least one engineered microcompartment protein operatively linked to one or more first regulatory elements configured to enable the expression of the at least one engineered microcompartment protein in one or more bacterial cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different engineered microcompartment proteins to form at least one microcompartment within the one or more bacterial cell.

The system additionally comprises at least one of:

the one or more bacterial cells capable of expressing the at least one first polynucleotide to provide an expressed engineered microcompartment protein herein described;

at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and

at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the protein non-native to the bacterial cell from the engineered microcompartment protein in the bacterial cell.

In the system, the at least one first polynucleotide, the at least one second polynucleotide the at least one protease and the one or more bacterial cells are combined or simultaneously or sequentially used in the methods to produce in a bacterial cell a protein non-native to the bacterial cell herein described.

In some embodiments, the at least one non-native protein in the engineered microcompartment protein is a non-native toxic protein capable of causing cell damage, and the engineered microcompartment protein is provided in the cell to shield cell from toxicity during intracellular production of a toxic non-native protein.

According to a fifth aspect, a method is described to produce a non-native protein in a bacterial cell comprising at least one engineered microcompartment protein herein described in which the target protein is a protein non-native to the cell. The method comprises introducing into the bacterial cell at least one second polynucleotide encoding the at least one protease capable of cleaving the at least one first protease cleavage site and/or the at least one second protease cleavage site within the engineered microcompartment protein. In the method, the at least one second polynucleotide is operably linked to one or more second regulatory elements configured to enable expression in the bacterial cell of the at least one protease. In the method, the introducing is performed to obtain the non-native protein from the engineered microcompartment protein upon cleaving of the at least one first protease cleavage site and/or the at least one second protease cleavage site by the at least one protease expressed in the bacterial cell.

According to a sixth aspect a system to produce a non-native protein from a bacterial cell comprising at least one engineered microcompartment protein herein described in which the target protein is a protein non-native to the cell, the system comprises

one or more bacterial cells comprising at least one engineered microcompartment protein herein described assembled with one or more same and/or different microcompartment proteins to form at least one engineered microcompartment within the cell.

The system also comprises

at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and

at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the one or more bacterial cells.

In the system, the one or more bacterial cells, the at least one second polynucleotide, and the at least one protease are used either simultaneously or sequentially in the methods to provide one or more non-native proteins in from one or more bacterial cell comprising an engineered microcompartment protein herein described.

In some embodiments, the at least one non-native protein in the engineered microcompartment protein is a non-native toxic protein capable of reacting with a native membrane substrate with a reaction resulting in a cell damage, and the engineered microcompartment protein is provided in the cell to shield cell from toxicity during intracellular production of a toxic non-native protein.

According to a seventh aspect, a vector is described comprising at least one polynucleotide encoding for an engineered microcompartment protein herein described alone or in combination with regulatory elements in accordance with the disclosure.

According to an eight aspect, a bacterial cell is described obtained by any one of the methods and/or with any one of the systems of the present disclosure.

According to a ninth aspect, a composition is described. The composition comprises the engineered microcompartment protein, the engineered microcompartment and/or the bacterial cell herein described.

Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments in connection with expression, production and/or purification in a bacterial cell of one or more proteins toxic to the bacterial cell or precursor thereof.

Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments to shield bacteria from toxicity during expression, production and/or purification of non-native toxic protein.

Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments in connection with expression, production and/or purification in a bacterial cell of one or more proteins degradable in the bacterial thereof.

Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be applied in several fields, including basic biology research, applied biology, bio-engineering, bio-energy, medical research, medical diagnostics, therapeutics, bio-fuels, and in additional fields where expression, production and/or purification in a bacterial cell of proteins which are degradable and/or cytotoxic to the bacterial cell and/or their precursors can be used.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings, incorporated herein by reference in its entirety and the description below. Other features, objects, and advantages will be apparent from the following description, and the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with detailed description and the examples, serve to explain the principles and implementations of the disclosure.

FIG. 1A illustrates a crystal structure of an exemplary encapsulin cage with 60 monomer units (one unit highlighted in red). The left panel shows a view from the outside on the five-fold symmetry axis. One pentamer is highlighted in cyan, with one monomer in red. The right panel shows a view to the inside of the shell, which is cut open in the middle and shown in a surface representation. FIG. 1B shows the T. maritima encapsulin monomer showing the A- and P-domains and the protruding E-loop. The monomer is colored in a rainbow scheme from the N terminus (blue) to the C terminus (red), the domains are named according to the homologous gp5 major capsid protein of the HK97 virus (PDB 1OHG). This figure is adapted from Sutter, M., et al., Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nature Structural & Molecular Biology, 2008. 15(9): p. 939-947.

FIG. 2 illustrates a BLOSUM62 matrix used for sequence alignment of proteins

FIG. 3 shows a pairwise alignment of an encapsulin from T. maritima (T. mar, SEQ ID NO: 45) and an encapsulin from M xanthus (M xan, SEQ ID NO: 46).

FIG. 4A shows the consensus sequence of exemplary encapsulins (SEQ ID NO: 132). FIG. 4B illustrates the segmentation of secondary structures of the encapsulins with regard to the consensus sequence.

FIG. 5 shows an exemplary encapsulin sequence from T. maritima (SEQ ID: 133).

FIG. 6 shows sequence alignment of 44 exemplary members from the PF04454 protein family and in particular, GI 122304915 (SEQ ID NO: 86), GI 123527077 (SEQ ID NO: 87), GI 123044215 (SEQ ID NO: 887), GI 123046337 (SEQ ID NO: 89), GI 81373191 (SEQ ID NO: 90), (SEQ ID NO: 91), (SEQ ID NO: 92), (SEQ ID NO: 93), (SEQ ID NO: 94), GI 81833634 (SEQ ID NO: 95), GI 74556699 (SEQ ID NO: 96), GI 81347829 (SEQ ID NO: 97), GI 81344013 (SEQ ID NO: 98), GI 123497228 (SEQ ID NO: 99), GI 501179632 (SEQ ID NO: 100), GI 502293893 (SEQ ID NO: 101), GI 501012501 (SEQ ID NO: 102), GI 506340687 (SEQ ID NO: 103), GI 502591318 (SEQ ID NO: 104), GI 501194893 (SEQ ID NO: 105), GI 490598858 (SEQ ID NO: 106), GI 752720587 (SEQ ID NO: 107), GI 501771872 (SEQ ID NO: 108), GI 501163578 (SEQ ID NO: 109), GI 501434203 (SEQ ID NO: 110), GI 501367709 (SEQ ID NO: 111), GI 505232787 (SEQ ID NO: 112), GI 501923113 (SEQ ID NO: 113), GI 502776253 (SEQ ID NO: 114), GI 502633921 (SEQ ID NO: 115), GI 496662878 (SEQ ID NO: 116), GI 500836508 (SEQ ID NO: 117), GI 501047338 (SEQ ID NO: 118), 4PT2_P (SEQ ID NO: 119), GI 501055150 (SEQ ID NO: 120), GI 501691096 (SEQ ID NO: 121), GI 494995233 (SEQ ID NO: 122), GI 501364857 (SEQ ID NO: 123), GI 502892820 (SEQ ID NO: 124), GI 527109103 (SEQ ID NO: 125), GI 501346422 (SEQ ID NO: 126), GI 501373147 (SEQ ID NO: 127), GI 501587999 (SEQ ID NO: 128), GI 500074236 (SEQ ID NO: 129), GI 501827525 (SEQ ID NO: 130), GI 521295581 (SEQ ID NO: 131)

FIG. 7 shows a schematic illustration of insertion sites for target proteins, protease cleavage sites and/or tags in embodiments herein described. In particular, FIG. 7A shows insertion sites of a target protein in engineered microcompartment herein described. FIG. 7B shows insertion sites of a target protein in engineered microcompartment herein described. FIG. 7B FIG. 7C and FIG. 7D shows insertion sites of a protease cleavage sites in engineered microcompartment herein described.

FIG. 8 shows in one embodiment an exemplary engineered microcompartment protein comprising Apidaecin 1a peptide fused to the N-terminus of an encapsulin protein from M xanthus through a TEV protease cleavage site and a linker region (SEQ ID: 134).

FIGS. 9A-B illustrate two exemplary systems for encapsulation of AMPS.

FIG. 10 illustrates a design of sense-control-release systems for P. aeruginosa. Panel A) Primary circuit strategy. LasR and AMP-encapsulin are produced from constitutive sigma70 promoters. 3OC12HSL-bound LasR drives expression of an ECF sigma factor (ECFsf), which in turn drives expression of the protease/lysis cassette as well as an anti-sigma factor. The anti-sigma factor inhibits the ECFsf and thus turns off expression of the protease/lysis cassette in a negative feedback loop. Panel B) Alternative circuit where 3OC12HSL-bound LasR drives expression of AMP-encapsulin and the ECFsf.

FIG. 11 illustrates the design of a protease-sensitive encapsulin.

FIG. 12 illustrates an exemplary testing of therapeutic delivery system efficacy in bacterial liquid culture.

FIG. 13 illustrates an exemplary testing of therapeutic delivery system efficacy in biofilm.

FIG. 14 illustrates an exemplary testing of therapeutic delivery system efficacy in in host tissue culture model

FIGS. 15A-C illustrate the design and sequences of the exemplary protease-sensitive AMP-encapsulin fusions(SEQ ID NO: 135-138).

FIG. 16 illustrate exemplary Ap-containing constructs as controls.

FIG. 17 shows in one embodiment expression of constructs pMCY124, pMCY125 and pMCY133 in comparison with control constructs.

FIG. 18 shows in one embodiment purification of constructs pMCY124, pMCY125 and pMCY133.

FIGS. 19A-C show in one embodiment TEV protease cleavage of the purified Ap-encapsulin fusions. All samples were analyzed by SDS-PAGE using both an any-kDa gel to analyze fragment >15 kDa (FIG. 19A) and a 16.5% Tris-Tricine gel to analyze fragments <15 kDa (FIG. 19B). Samples were also analyzed by Western blot using an anti-TEV site antibody (FIG. 19C). Arrows on the gels above show digested fragments.

FIG. 20A illustrates gene cassettes expressing up to 4 Ap peptides fused to a single Encapsulin construct. FIG. 20B shows the expression of the gene cassettes in FIG. 19A with the samples resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue. Red arrows denote predicted location of the expressed protein.

FIG. 21A illustrates gene cassettes expressing up to 3 HB peptides fused to a single Encapsulin construct as well as a control of HB fused to thioredoxin (Trx). FIG. 21B shows the expression of the gene cassettes in FIG. 21A with the samples resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue. Red arrows denote predicted location of the expressed protein.

FIG. 22A shows a schematic illustration of gene constructs for HBCM2 fusions with engineered Enc. TEV denotes TEV protease recognition sites. 6× His denotes a hexa-histidine tag for purification. Linkers shown in light gray are described in FIG. 22B. FIG. 22B shows a table reporting the amino acid sequences of HB-Enc constructs (SEQ ID NO: 48-54) and fusions of HB peptide to other common carrier proteins (SEQ ID NO: 55-58). Plain text, indicates Enc or carrier protein sequence, Italics, linker; Bold, TEV recognition site; Bold underlined, HBCM2; Italics underlined, His-tag.

FIG. 23A shows images illustrating the results of experiments to detect the expression of the HB fusion constructs in FIGS. 22A-B in C43(DE3) E. coli from a T7 IPTG inducible promoter. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue (top) or blotted to a PVDF membrane and probed with mouse anti-His₆ primary antibody and rabbit anti-mouse HRP conjugated secondary antibody (bottom). T denotes the total cell lysate, while S denotes the soluble fraction. Arrows denote expected size of the expressed protein. FIG. 23B shows a chart illustrating the final OD₆₀₀ of the C43(DE3) E. coli culture after overnight induction at 18° C.

FIG. 24 shows an image of a gel including purified HB-Enc fusion proteins with or without TEV protease digestion at 4° C. overnight. Samples were resolved on a 16.5% Tris-Tricine gel and stained with Commassie blue. In the bottom portion a table shows the normalized ratio of HB released/HB-Enc fusion based on densitometry, assuming the HB/HB-Enc ratio for HB-EncK71^(TEV)K138^(TEV-His) is 1.

FIG. 25 shows an image of a gel including purified HB-Enc fusion proteins with a GT linker replacing the flexible G₄T linker between the HB peptide and the N-terminus of Enc. Fusions were digested with or without TEV protease at 4° C. overnight and samples were resolved on a 16.5% Tris-Tricine gel and stained with Commassie blue.

FIG. 26 shows an image illustrating the results of a Native PAGE analysis of the HB-Enc constructs. Samples were resolved on an any-kDa Native PAGE gel and stained with Coomassie blue. Arrows denote the majority species. High molecular weight (MW) species are located at the top of the gel in the well area. Low MW species enter the gel and migrate lower into the gel.

FIG. 27 shows charts illustrating size exclusion chromatography traces of absorbance at 280 nm for a cage-forming construct (Ap-EncK138^(TEV-His)) and a non-cage forming construct (Ap-EncK71^(TEV)K138^(TEV-His)). Dashed line denotes the retention time of a 670 kDa standard. The void volume is labeled. Cage-forming, high MW species is denoted by a black arrow, while the non-cage-forming, low MW species is denoted by a white arrow.

FIG. 28 shows TEM images of cage-forming (EncK138^(His), HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), HB-EncV57^(TEV)K138^(TEV-His)) and non-cage forming (HB-EncK71^(TEV)K138^(TEV-His), HB-EncK71^(TEV)) HB-Enc constructs. Scale bar is 100 nm.

FIG. 29 shows an image illustrating Native PAGE analysis and TEM images of Ap-Enc fusions: Ap-EncK138^(TEV-His) and Ap-EncK71^(TEV)K138^(TEV-His). Black and white arrows denote high and low MW species, respectively. Scale bar on TEM images is 100 nm.

FIG. 30 shows results of experiments illustrating protease sensitivity of exemplary HB-Enc constructs in cell lysate. C43(DE3) E. coli expressing the constructs were either 1) lysed in the presence of BPER-II and lysozyme; 2) lysed in the absence of BPER-II by French pressure lysis; or 3) lysed in the absence of BPER-II by French pressure lysis and then incubated at 4° C. overnight. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue (top) or blotted to a PVDF membrane and probed with mouse anti-His₆ primary antibody and rabbit anti-mouse HRP conjugated secondary antibody (bottom). Black arrows denote size of full-length fusions. White arrows denote sizes of proteolysis products.

FIG. 31 Panel A shows anti-bacterial growth inhibition assays against E. coli BL21(DE3) for TEV-digested HB-EncK71^(TEV)K138^(TEV) and negative controls of undigested HB-EncK71^(TEV)K138^(TEV) alone and TEV protease alone. Only TEV-digested HB-EncK71^(TEV)K138^(TEV) has activity. FIG. 31 Panel B shows inhibition assays for various concentrations of chemically synthesized M-HBCM2-TEV peptide. The peptide has an MIC <5 μg/mL, consistent with native HBCM2 peptide.

FIG. 32 shows charts illustrating the results of anti-bacterial growth inhibition assays against E. coli BL21(DE3) for various concentrations of chemically synthesized native AP peptide (FIG. 32 Panel A) and M-AP-TEV peptide (FIG. 32 Panel B). Data shows M-AP-TEV peptide is not active compared to native AP peptide.

FIG. 33A shows expression of the HB fusion constructs in FIGS. 22A-B in TOP10 E. coli from a pBAD arabinose inducible promoter. Cells were induced with either 40 μM or 10 mM arabinose. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. T denotes the total cell lysate, while S denotes the soluble fraction. FIG. 33B shows comparison of full-length HB fusions expressed in either C43(DE3) cells (C43) or BL21(DE3) cells (BL21) versus HB fusions expressed in TOP10 cells from a pBAD promoter (Ara). In both FIGS. 33A and B, black arrows denote expected size of the expressed protein, while white arrows denote truncation products. FIG. 33C shows the final OD₆₀₀ of the TOP10 E. coli cultures after 4-5 h induction at 37° C.

FIG. 34 shows results of experiments directed to illustrate expression and protease sensitivity of exemplary constructs HB-Trx, HB-SUMO, HB-GST, and HB-MBP. FIG. 34 Panel A shows an image of a gel illustrating expression of the HB-Trx, HB-SUMO, HB-GST, and HB-MBP constructs in FIGS. 22A-B in BL21(DE3) E. coli from a T7 IPTG inducible promoter. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. T denotes the total cell lysate, while S denotes the soluble fraction. FIG. 34 Panel B shows an image of a gel illustrating protease sensitivity of the constructs in cell lysate. BL21(DE3) E. coli expressing the constructs were either 1) lysed in the presence of BPER-II and lysozyme; 2) lysed in the absence of BPER-II by French pressure lysis; or 3) lysed in the absence of BPER-II by French pressure lysis and then incubated at 4° C. overnight. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. For both Panels A and B, black arrows denote size of full-length fusions.

FIG. 35 shows an image of a gel illustrating isolation of M-Ap-TEV peptide following TEV protease cleavage of Ap-EncK71^(TEV)K138^(TEV-His). Samples were collected before and after cleavage of Ap-EncK71^(TEV)K138^(TEV-His) and peptide was collected in the filtrate after centrifugation on a 10 kDa molecular weight cutoff centrifugal filter. Samples were resolved on a 16.5% Tris-Tricine SDS-PAGE gel and stained with Commassie blue.

DETAILED DESCRIPTION

Provided herein are engineered microcompartment proteins and related engineered microcompartments, bacterial cells, compositions, methods and systems that can be used in several embodiments for expression, production and/or purification in a bacterial cell of proteins non-native to the bacterial cell.

The term “microcompartment” or “bacterial microcompartment” as used herein indicated organelles within a bacterial cell in which a protein shell encloses enzymes and other proteins. Microcompartments are typically about 40-200 nanometers in diameter and are entirely made of proteins in which the shell functions like a membrane, as it is selectively permeable. Exemplary microcompartments are described in application Ser. No. 15/178,454 filed on Jun. 6, 2016 and published on Dec. 15, 2016 with publication number US206/0362697 incorporated herein by reference in its entirety.

In embodiments herein described, the microcompartments are encapsulin microcompartments and the related microcompartment proteins are encapsulins or encapsulin like

The term “encapsulin” or “encapsulin-like” as used herein indicates proteins that are capable of self-assembling in a bacterial cell to form a microcompartment in which interior molecules (e.g., DNA, RNA, protein) can be encaged. In some instances encapsulin proteins can be native to the bacterial cells where they are expressed.

Accordingly, the wording “encapsulin-like microcompartments” or “cage” or “BMC” as used herein refers to organelles produced, and in particular possibly natively produced, by bacteria or viruses to organize and sequester biological molecules, such as DNA, RNA, or protein in a bacterial cell within the confines of a protein shell. Accordingly encapsulin microcompartment can be native or non-native to the cells where produced as would be understood by a skilled person. The encapsulin microcompartments typically have pseudo-icosahedral structures that can be 10 to 400 nm in diameter with a thickness of 20-30 Å [1]. Encapsulin-like protein protomers assemble into pentameric and/or hexameric shapes that further assemble to form the icosahedral microcompartment where the pentagons form the vertices and the hexagons form the flat facets of the compartment. All compartments have a total 20 T triangular faces that are formed from 12 pentagons and 10(T-1) hexagons. T is defined as the triangulation number, which can be any non-negative integer that fits T=ĥ2+k̂2+hk, where h and k are also non-negative integers (e.g., T can be 1, 3, 4, 7, etc.). Compartments with different numbers of protomers and T values can result in different sized and shaped compartments.

Encapsulin microcompartments typically comprise a microcompartment protein or shell protein forming the shell of the microcompartment and one or more interior proteins identifiable by a skilled person. An encapsulin shell protein typically has three common conserved domains: a peripheral domain (P-domain), an axial domain (A-domain), and an elongated loop (E-loop). Common examples of encapsulin shell proteins include encapsulins Enc A from Thermotoga maritima and Myxococcus xanthus, and Pfv from P. furiosus. [1].

Common examples of encapsulins interior proteins comprise EncB, EncC, and EncD from M xanthus wherein EncB and EncC are ferritin-like proteins that are thought to bind and sequester iron, while EncD has unknown function. Together they are thought to sequester iron under oxidative stress conditions [McHugh, 2014]. Other interior exemplary encapsulins comprise the dye-decoloring peroxidase DyP protein from T maritima as an internal protein.

Encapsulin-like microcompartments can be of bacterial or viral origin. Exemplary encapsulin-like proteins of bacterial origin include encapsulin or virus-like compartments from Thermotoga maritima (T=1, 24 nm diameter), Pyrococcus furiosus (T=3, 31 nm diameter), and Myxococcus xanthus (T=3, 31 nm diameter). Exemplary encapsulin-like proteins of viral origin include HK97 phage capsid (T=7, 66 nm diameter).

A representative example of encapsulin proteins is provided by Thermotoga maritima encapsulin. In T. maritima encapsulin microcompartment, sixty monomers of T. maritima encapsulin assemble into a spherical superstructure with icosahedral T=1 symmetry, a diameter of 230-240 Å and a thickness of 20-25 Å. [2] as shown in FIG. 1A. The peripheral domain (P-domain), axial domain (A-domain), and elongated loop (E-loop) of Enc A of T. maritima are schematically shown in FIG. 1B).

Microcompartments from bacteria can be isolated and detected by methods and systems exemplified herein (see Example 28) and by additional methods and systems identifiable by a skilled person upon review of the present disclosure.

In embodiments herein described, microcompartment proteins comprised within engineered microcompartment proteins of the disclosure are encapsulin proteins having sequence

X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇—X₁₈—X₁₈—X₂₀—X₂₁—X₂₂—X₂₃—X₂₄—X₂₅—X₂₆—X₂₇—X₂₈—X₂₉—X₃₀ X₃₁—X₃₂—X₃₃ X₃₄—X₃₅—X₃₆—X₃₇—X₃₈—X₃₉—X₄₀—X₄₁—X₄₂—X₄₃—X₄₄—X₄₅—X₄₆—X₄₇—X48—X₄₉—X₅₀—X51—X₅₂—X₅₃—X₅₄—X₅₅—X56—X₅₇—X₅₈—X₅₉—X₆₀—X₆₀—X₆₁—X₆₂—X₆₃—X₆₄—X₆₅—X₆₆—X₆₇—X₆₈—X₆₉—X₇₀—X₇₁—X₇₂—X₇₃—X₇₄—X₇₅—X₇₆—X₇₇—X₇₈—X₇₉—X₈₀—X₈₁—X₈₂—X₈₃—X₈₄—X₈₅—X₈₆—X₈₈—X₈₉—X₉₀—X₉₁—X₉₂—X₉₃—X₉₄—X₉₅—X₉₆—X₉₇—X₉₈—X₉₉—X₁₀₀—X₁₀₁—X₁₀₂—X₁₀₃—X₁₀₄—X₁₀₅—X₁₀₆—X₁₀₇—X₁₀₈—X₁₀₉—X₁₁₀—X₁₁₁—X₁₁₂—X₁₁₃—X₁₁₄—X₁₁₅—X₁₁₆—X₁₁₇—X₁₁₈—X₁₁₉—X₁₂₀—X₁₂₁—X₁₂₂—X₁₂₃—X₁₂₄—X₁₂₅—X₁₂₆—X₁₂₇—X₁₂₈—X₁₂₉—X₁₃₀—X₁₃₁—X₁₃₂—X₁₃₃—X₁₃₄—X₁₃₅—X₁₃₆—X₁₃₇—X₁₃₈—X₁₃₉—X₁₄₀—X₁₄₁—X₁₄₂—X₁₄₃—X₁₄₄—X₁₄₅—X₁₄₆—X₁₄₇—X₁₄₈—X₁₄₉—X₁₅₀—X₁₅₁—X₁₅₂—X₁₅₃—X₁₅₄—X₁₅₅—X₁₅₆—X₁₅₇—X₁₅₈—X₁₅₉—X₁₆₀—X₁₆₁—X₁₆₂—X₁₆₃—X₁₆₄—X₁₆₅—X₁₆₆—X₁₆₇—X₁₆₈—X₁₆₉—X₁₇₀—X₁₇₁—X₁₇₂X₁₇₃—X₁₇₄—X₁₇₅—X₁₇₆—X₁₇₇—X₁₇₈—X₁₇₉—X₁₈₀—X₁₈₁—X₁₈₂—X₁₈₃—X₁₈₄—X₁₈₅—X₁₈₆—X₁₈₇—X₁₈₈—X₁₈₉—X₁₉₀—X₁₉₁—X₁₉₂—X₁₉₃—X₁₉₄—X₁₉₅—X₁₉₆—X₁₉₇—X₁₉₈—X₁₉₉—X₂₀₀—X₂₀₁—X₂₀₂—X₂₀₃—X₂₀₄—X₂₀₅—X₂₀₆—X₂₀₇—X₂₀₈—X₂₀₉—X₂₁₀—X₂₁₁—X₂₁₂—X₂₁₃—X₂₁₄—X₂₁₅—X₂₁₆—X₂₁₇—X₂₁₈—X₂₁₉—X₂₂₀—X₂₂₁—X₂₂₂—X₂₂₃—X₂₂₄—X₂₂₅—X₂₂₆—X₂₂₇—X₂₂₈—X₂₂₉—X₂₃₀—X₂₃₁—X₂₃₂—X₂₃₃—X₂₃₄—X₂₃₅—X₂₃₆—X₂₃₇—X₂₃₈—X₂₃₉—X₂₄₀—X₂₄₁—X₂₄₂—X₂₄₃ —X₂₄₄—X₂₄₅—X₂₄₆—X₂₄₇—X₂₄₈—X₂₄₉ X₂₅₀—X₂₅₁—X₂₅₂—X₂₅₃ (SEQ ID NO: 1).

in which X₁ is M, X₂ is D, X₃ is N, X₄ is L, X₅ is K, X₆ is R, X₇ is E, X₈ is L, X₉ is A, X₁₀ is P, X₁₁ is L, X₁₂ is T, X₁₃ is E, X₁₄ is E, X₁₅ is A, X₁₆ is W, X₁₇ is A, X₁₈ is E, X₁₉ is I, X₂₀ is D, X₂₁ is E, X₂₂ is E, X₂₃ is A, X₂₄ is R, X₂₅ is E, X₂₆ is T, X₂₇ is A, X₂₈ is K, X₂₉ is R, X₃₀ is H, X₃₁ is L, X₃₂ is A, X₃₃ is G, X₃₄ is R, X₃₅ is R, X₃₆ 1S V, X₃₇ 1S V, X_(3g) is D, X₃₉ is V, X₄₀ is E, X₄₁ is G, X₄₂ is P, X₄₃ is L, X₄₄ is G, X₄₅ 1S W, X₄₆ is G, X₄₇ is Y, X₄₈ is S, X₄₉ is A, X₅₀ is V, X₅₁ is P, X₅₂ is L, X₅₃ is G, X₅₄ is R, X₅₅ is L, X₅₆ is E, X₅₇ is E, X₅₈ is I, X₅₉ is E, X₆₀ is G, X₆₁ is P, X₆₂ is A, X₆₃ is E, X₆₄ is G, X₆₅ is V, X₆₆ is Q, X₆₇ is A, X₆₈ is G, X₆₉ is V, X₇₀ is R, X₇₁ is Q, X₇₂ is V, X₇₃ is L, X₇₄ is P, X₇₅ is L, X₇₆ is P, X₇₇ is E, X₇₈ is L, X₇₉ is R, X_(g0) is V, X₈₁ is P, X₈₂ is F, X₈₃ is T, X₈₄ is L, X₈₅ is S, X₈₆ is R, X₈₇ is R, X₈₈ is D, X₈₉ is L, X₉₀ is D, X₉₁ is A, X₉₂ is V, X₉₃ is E, X₉₄ is R, X₉₅ is G, X₉₆ is A, X₉₇ is K, X₉₈ is D, X₉₉ is L, X₁₀₀ is D, X₁₀₁ is L, X₁₀₂ is S, X₁₀₃ is P, X₁₀₄ is V, X₁₀₅ is VA, X₁₀₆ is E, X₁₀₇ is A, X₁₀₈ is A, X₁₀₉ is R, X₁₁₀ is L, X₁₁₁ is L, X₁₁₂ is A, X₁₁₃ is R, X₁₁₄ is A, X₁₁₅ is E, X₁₁₆ is D, X₁₁₇ is R, X₁₁₈ is L, X₁₁₉ is I, X₁₂₀ is F, X₁₂₁ is N, X₁₂₂ is G, X₁₂₃ is Y, X₁₂₄ is A, X₁₂₅ is E, X₁₂₆ is A, X₁₂₇ is G, X₁₂₈ is I, X₁₂₉ is E, X₁₃₀ is G, X₁₃₁ is L, X₁₃₂ is L, X₁₃₃ is N, X₁₃₄ is A, X₁₃₅ 1S S, X₁₃₆ is G, X₁₃₇ is N, _(X138) is L, X₁₃₉ is K, X₁₄₀ is L, X₁₄₁ is P, X₁₄₂ is L, X₁₄₃ is S, X₁₄₄ is A, X₁₄₅ is D, X₁₄₆ is P, X₁₄₇ is G, X₁₄₈ is D, X₁₄₉ is I, X₁₅₀ is P, X₁₅₁ is D, X₁₅₂ is A, X₁₅₃ is I, X₁₅₄ is A, X₁₅₅ is E, X₁₅₆ is A, X₁₅₇ is L, X₁₅₈ is T, X₁₅₉ is K, X₁₆₀ is L, X₁₆₁ is R, X₁₆₂ is E, X₁₆₃ is A, X₁₆₄ is G, X₁₆₅ is V, X₁₆₆ is E, X₁₆₇ is G, X₁₆₈ is P, X₁₆₉ is Y, X₁₇₀ is A, X₁₇₁ is L, X₁₇₂ is V, X₁₇₃ is L, X₁₇₄ is S, X₁₇₅ is P, X₁₇₆ is D, X₁₇₇ is L, X₁₇₈ is Y, X₁₇₉ is T, X₁₈₀ is A, X₁₈₁ is L, X₁₈₂ is F, X₁₈₃ is R, X₁₈₄ is V, X₁₈₅ is Y, X₁₈₆ is D, X₁₈₇ is G, X₁₈₈ is T , X₁₈₉is G, X₁₉₀ is Y, X₁₉₁ is P, X₁₉₂ is E, X₁₉₃ is I, X₁₉₄ is E, X₁₉₅ is H, X₁₉₆ is I, X₁₉₇ is K, X₁₉₈ is E, X₁₉₉ is L, X₂₀₀ is V, X₂₀₁ is D, X₂₀₂ is G, X₂₀₃ is G, X₂₀₄ is V, X₂₀₅ is I, X₂₀₆ is W, X₂₀₇ is A, X₂₀₈ is P, X₂₀₉ is A, X₂₁₀ is L, X₂₁₁ is D, X₂₁₂ is G, X₂₁₃ is G, X₂₁₄ is A, X₂₁₅ 1S V, X₂₁₆ is L, X₂₁₇ 1S V, X₂₁₈ is S, X₂₁₉ is T, X₂₂₀ is R, X₂₂₁ is G, X₂₂₂ is G, X₂₂₃ is D, X₂₂₄ is F, X₂₂₅ is D, X₂₂₆ is L, X₂₂₇ is T, X₂₂₈ is L, X₂₂₉ is G, X₂₃₀ is Q, X₂₃₁ is D, X₂₃₂ is L, X₂₃₃ is S, X₂₃₄ is I, X₂₃₅ is G, X₂₃₆ is Y, X₂₃₇ is L, X₂₃₈ is S, X₂₃₉ is H, X₂₄₀ is D, X₂₄₁ is A, X₂₄₂ is D, X₂₄₃ is N, X₂₄₄ is V, X₂₄₅ is E, X₂₄₆ is L, X₂₄₇ is F, X₂₄₈ is L, X₂₄₉ is T, X₂₅₀ is E, X₂₅₁ 1S S, X₂₅₂ is F, X₂₅₃ is T (SEQ ID NO: 1) or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:l.

In preferred embodiments, the engineered microcompartment protein of the disclosure, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 30% sequence identity, a 40% sequence identity, at least a 50% sequence identity, or more preferably at least a 60% sequence identity or greater with SEQ ID NO: 1 or at least 40% sequence similarity, preferably at least 50% sequence similarity, at least 60% sequence similarity or a greater similarity to SEQ ID NO:1.

The term “sequence identity” indicates the amount of characters that are identical between two different sequences following alignment of the sequences. The percent identity is calculated from the number of exact character matches divided by the total number of aligned characters, including gaps, multiplied by 100%.

The term “sequence similarity” indicates the amount of characters that are similar between two different sequences following alignment of the sequences. Different from the “identity”, which refers to an exact match between two nucleotides or amino acids, similarity refers to a resemblance between two residues that is greater than one would expect at random and provides a predictable structure. Amino acids are considered similar if they have a positive value in a substitution matrix, such as BLOSUM-62 (FIG. 2). BLOSUM-62 is a probability matrix based on observed substitutions found in a broad sampling highly aligned sequences [3] and is used in the BLAST alignment tool. Percent similarity is calculated from the number of similar amino acid matches (based on the substitution matrix) divided by the total number of aligned characters, including gaps, multiplied by 100%.

Sequence identity and sequence similarity can be detected by commonly used searching programs, like BLAST, PSI-BLAST [4], SSEARCH [5] [6], FASTA [7] and the HMMER3 [8] which can produce accurate statistical estimates of protein sequences that share sequence identity, similarity and also have similar structures.

The identity or similarity between sequences is typically measured by a process that comprises the steps of aligning two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, characters similar or identical between the two aligned sequences, and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity or identity result is expressed as a percentage.

An exemplary alignment is illustrated in FIG. 3 which shows a pairwise alignment of encapsulin from T. maritima compared to encapsulin from M xanthus. In the representative example of FIG. 3 a total of 279 residues are aligned. 64 of the residues have an exact match between the two sequences which is denoted as the given letter symbol in the line between the two sequences. In this case, the percent identity is 64/279×100%=23% identity. 122 of the residues (including the 64 exact identities) have positive values based on the BLOSUM-62 similarity matrix, which are denoted with either a +symbol or the letter symbol in the line between the two sequences. In this case, the percent similarity is 122/279×100%=43%.

In embodiments herein described, an encapsulin protein which either has SEQ ID NO: 1 or a sequence of the disclosure with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises a P-Domain divided into a first fragment, a second fragment and a third fragment, an E-Loop and an A-Domain, each having an N-terminus and a C-terminus.

The term the “amino terminus” or “N-terminus” indicate the amino acid residue of a linear polypeptide chain at one of the extremities of the linear polypeptide chain which, when not involved in a peptide bond, presents an amino group. The term the “carboxyl terminus” “C-terminus” indicate the amino acid residue of a linear polypeptide chain at one of the extremities of the linear polypeptide chain which, when not involved in a peptide bond, presents a carboxyl group. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NH2-group). An N terminus or a C-terminus of a polypeptide is typically comprised within a “tail” of the protein which indicates a segment or fragment at the related end of the protein.

The term “fragment’ or “segment” as related to the protein indicates any continuous part of a protein sequence from single amino acid up to the full protein associated to an identifiable structure within the protein. An “identifiable structure” in the sense of the disclosure indicates a spatial arrangement of the primary structure or portions thereof which can be detected by techniques such as crystallography, hydrophobicity analysis or additional techniques known by a skilled person. In some instances, a protein segment or fragment can comprise one or more secondary structures of the protein.

The “secondary structure” of a protein refers to local sub-structures with a repeating geometry identifiable within crystal structure of the protein, circular dichroism or by additional techniques identifiable by a skilled person. In some instances, a secondary structure of a protein can be identified by the patterns of hydrogen bonds between backbone amino and carboxyl groups. Secondary structures can also be defined based on a regular, repeating, geometry, being constrained to approximate values of the dihedral angles ψ and φ of the amino acids in the secondary structure unit on the Ramachandran plot. Two main types of secondary structure are the alpha helix αnd the beta strand or beta sheets as will be identifiable by a skilled person. Both the alpha helix and the beta sheet represent a way of establishing non-covalent hydrogen bonds between constituents of the peptide backbone. Secondary structure formation can be promoted by formation of hydrogen bonds between backbone atoms. Amino acids that can minimize formation of a secondary structure by destabilizing the structure of the hydrogen bonding interactions are referred to as secondary structure breakers. Amino acids that can promote formation of a secondary structure by stabilizing formation of hydrogen bonding interactions are referred to as structure makers.

Several sequential secondary structures may form a “supersecondary unit” or “structural motif.” A “supersecondary unit” or “structural motif” indicates a segment of the protein that forms an identifiable three-dimensional structure formed by adjacent secondary structure elements optionally linked by unstructured protein regions. In structural motifs the secondary structures are typically comprised with a same orientation one with respect to another. In particular some structural motifs (e.g. zinc fingers, a Greek key or helix—turn helix) are conserved in different proteins as will be understood by a skilled person.

The “tertiary structure” of a protein refers to the three-dimensional structure of a protein, stabilized by non-covalent interactions among non-adjacent segments of the protein and optionally by one or more additional compounds or ions interacting through covalent or non-covalent interactions with one or more segments of the proteins. Exemplary non-covalent interactions stabilizing the three dimensional structure of the proteins comprise non-specific hydrophobic interactions, burial of hydrophobic residues from water, specific tertiary interactions, such as salt bridges, hydrogen bonds, the tight packing of side chains, chelation and disulfide bonds and additional interactions identifiable by a skilled person. Exemplary covalent interactions among compounds or ions and segments of the protein comprise, N-linked glycosylation, cytochrome C heme attachment and additional interaction identifiable by a skilled person.

In embodiments herein described, the first fragment of the P-Domain of an encapsulin protein of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus C-terminus direction, a 4 to 26 residues alpha helix αl, followed by a 0 to 22 residues first non-structured region (e.g. forming a loop), linked to a 4 to 11 residues alpha helix α2, linked to a 3 to 9 residues beta strand β1, linked to a 3 to 13 residues second non-structured region. In particular, in embodiments, where the encapsulin protein has SEQ ID NO: 1, the first fragment of the P-Domain can be formed by residues X2 to X46. A representative example of the structure of a first fragment of the P-domain according to these embodiments is provided by the first segment of the P-domain of the encapsulin shell protein EnCA illustrated in FIG. 1B. As shown, the first segment of the P-Domain in the representative EncA of FIG. 1B consists of a mixed α/β structure, contains the N terminus and is fragmented with regard to primary sequence (FIG. 1B, orange secondary structure bars). The first fragment or segment of the P-domains contains two alpha helices α1, α2 and one beta strand β1.

In embodiments herein described, the second fragment of the P-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus to C-terminus direction a 9 to 15 residues beta strand β4, linked to a 6 to 15 residues alpha helix α3, linked to a 0 to 10 residues first non-structured region, linked to a 18 to 29 residues alpha helix α4, linked to a 9 to 21 residues second non-structured region. In particular, in embodiments, where the encapsulin protein has SEQ ID NO: 1, the second fragment of the P-Domain can be formed by residues X75 to X130. A representative example of the structure of a second fragment of the P-domain according to these embodiments is provided by the second segment of the P-domain of the encapsulin shell protein EnCA illustrated in FIG. 1B. As shown, the second segment of the P-Domain in the representative EncA of FIG. 1B consists of the second segment of the P-domain contains one beta strand β4 and two alpha helices α3 and α4.

In embodiments herein described, the third fragment of the P-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus to C-terminus direction, a 4 to 10 residues beta strand β9, linked to by a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand β10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand β11. In particular, embodiments, where the microcompartment protein has SEQ ID NO: 1, the third fragment of the P-Domain can be formed by residues X221 to X253. A representative example of the structure of a second fragment of the P-domain according to these embodiments is provided by the third segment of the P-domain of the encapsulin shell protein EncA illustrated in FIG. 1B. As shown, the third segment of the P-Domain in the representative EncA of FIG. 1B consists of the third segment of the P-domain contains three beta strands β9, β10, and β11. A conserved hydrophobic core is located between the helical and β-sheet regions.

In the engineered microcompartment protein, the E-Loop of the encapsulin protein of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in an N-terminus to C-terminus direction, a 8 to 16 residues beta strand β2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand β3, linked to a 0 to 6 residues second non-structured region. In particular, in embodiments, where the microcompartment protein has SEQ ID NO: 1, the E-Loop can be formed by residues X47 to X74. A representative example of the structure of an E-Loops according to these embodiments is provided by the E-Loop of the encapsulin shell protein EnCA is illustrated in FIG. 1B. In the illustration of FIG. 1B, the E-loop adopts a flexible loop conformation and is responsible for the formation of contacts between the two-fold symmetry-related subunits by providing a strand that completes a β-sheet formed by both subunits. The E-loop contains two β strands: β2 and β3.

In the engineered microcompartment protein, the A-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in an N-terminus to C-terminus direction a 0 to 8 residues beta strand β5, linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix α5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand β6, a 9 to 16 residues alpha helix α6, a 1 to 24 third non-structured region, linked to a 0 to 16 residues alpha helix α7, linked to a 0 to 8 residues fourth non-structured region, linked to a 1 to 10 residues beta strand β7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand β8, linked to a 2 to 12 residues sixth non-structured region In in particular, in embodiments, where the microcompartment protein has SEQ ID NO: 1, the A-Domain can be formed by residues X131 to X220. A representative example of the structure of an A-Domain according to these embodiments provided by the A-Domain of the encapsulin shell protein EnCA is illustrated in FIG. 1B. In the illustration of FIG. 1B, the A-domain forms a compact structure consisting of three helical segments and a five-stranded β-sheet. The A-domain also contains the C terminus of the encapsulin shell protein. This domain has few connections to the rest of the monomer and mediates the contacts of the five-fold symmetry interface. The A-domain contains four beta strands β5, β6, β7, β8 and three alpha helices α5, α6, α7.

In an encapsulin of an engineered microcompartment protein herein described, each P-domain, A-domain and E-loop of the engineered microcompartment proteins of the current disclosure has a N-terminus and a C-terminus, and the P-domain, A-domain and E-loop are arranged together in a configuration comprising in a direction N-terminus to C-terminus the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain. In particular, in encapsulin protein used for constructing engineered microcompartment proteins herein described the C-terminus of the first fragment of P-domain is covalently attached to the N-terminus of the E-loop, the C-terminus of the E-loop is covalently linked to the N-terminus of the second fragment of the P-domain, the C-terminus of the second fragment of the P-domain is covalently attached to the N-terminus of the A-domain, and the C-terminus of the A-domain is covalently attached to the N-terminus of the third fragment of the P-domain (see configuration of the representative EncA of FIG. 1B).

In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X2 to X46, X75 to X130 and X221 to X253 form a P-domain, in which residues X2 to X46 form a first fragment of the P-domain, X75 to X130 form a second fragment of the P-domain and X221 to X253 form a third fragment of the P-domain.

In particular, in some embodiments, one insertion site can be located within the loop region of the E-loop between β2 and β3, comprising X57 to X65, another insertion site can be located within β3 of the E-loop, comprising X66 to X74, and/or another insertion site can be located in the flexible region between the N-terminus of the A-domain and α5, including β3, comprising X132 to X144, as will be understood by a skilled person.

In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X47 to X74 form an E-loop.

In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X131 to X220 form an A-domain, residues X221 to X253 form a third fragment of the P-domain

Various embodiments of encapsulin proteins in the sense of the disclosure that can be used for constructing an engineered microcompartment herein described, have a consensus sequence of SEQ ID NO:2 reported below. The amino acids highlighted in bold in SEQ ID NO: 2 and in other sequences herein described (see e.g. T. maritima encapsulin sequence (FIG. 5) indicates either identical or highly conserved among the different encapsulins used for constructing an engineered microcompartment herein described, unless otherwise indicated. The wording “highly conserved” indicates identical/conserved amino acids passing a 3.0 bit conservation setting based on pfam alignment tool for all 44 sequences in pfam 04454 (see https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi at the filing date of the present disclosure).

(SEQ ID NO: 2) MDNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WGYSAVPLGRL 55 EEIEGPAEGVQ AGVRQVLPLPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK 110 LARAEDRLIFN GYAEAGIEGLL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV 165 EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR 220 GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT

FIG. 4 shows an additional representation of consensus sequences SEQ ID NO: 2 with complete indication of the related P-Domain, E-Loop and E Domain (FIG. 4A) as well as a schematic representation of the related configuration in the encapsulin sequence (FIG. 4B).

In particular, consensus sequence SEQ ID NO: 2 contains 253 amino acids forming a 3-D configuration having a P-domain, A-domain and an E-loop. In particular, in consensus sequence SEQ ID NO: 2, residues DNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WG (SEQ ID NO:3) provide the consensus for the first fragment of the P-domain containing two alpha helices α1, α2 and one beta strand β1, in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of the fragment. Residues YSAVPLGRL EEIEGPAEGVQ AGVRQVLP (SEQ ID NO: 4) provide the consensus for the E-loop containing two β strands: β2 and β3. Residues LPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK LARAEDRLIFN GYAEAGIEG (SEQ ID NO:5) provide the consensus for the second fragment of the P-domain containing one beta strand β4 and two alpha helices α3 and α4 in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of the fragment. Residues LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6) provide the consensus for the A-domain containing four beta strands β5, β6, β7, β8 and three alpha helices α5, α6, α7 and residues GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT (SEQ ID NO:7) form the third fragment of the P-domain containing three beta strands β9, β10, and β11 in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of each respective fragment.

A skilled person will be able to identify encapsulin proteins in the sense of the disclosure from the consensus sequence SEQ ID NO: 2 following one or more sequence alignments upon reading of the present disclosure.

In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described have at least a 30% sequence identity, at least a 40% sequence identity, at least a 50% sequence identity, or at least a 60% sequence identity or greater with the encapsulin protein of SEQ ID NO: 2.

In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described have at least a 50% sequence similarity, at least a 60% sequence similarity or a greater similarity with the encapsulin shell protein of SEQ ID NO: 2.

In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the E-Loop which following sequence alignment provide a consensus sequence YSAVPLGRL EEIEGPAEGVQ AGVRQVLP (SEQ ID NO:4) In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise an E-loop having a primary sequence of at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 4. In some of these embodiments the E loop sequences of engineered microcompartment proteins herein described comprise a V57 residue and/or a D60 residue such as 3KDT (T. maritima) YAAHPLGEVEVLSDENEVVKWGLRKSLP (SEQ ID NO: 59), GI 501012501 YTVVPEGRLKKIEDNPGNVCTGMYQVKP (SEQ ID NO: 60), GI 502591318 YAAVNTGELRPIDDTPEDVDMKLRQVQP (SEQ ID NO: 61), GI 501771872 YAAVNTGRRTALEDKAEGASIFQRQVLP (SEQ ID NO: 62), and GI 501367709 FSALGTGHVSRVAADTPGVEALQRHVVR (SEQ ID NO: 63).

In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the A-domain which following sequence alignment provide a consensus sequence LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6). In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise A-domain having a primary sequence of at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 6. In some of these embodiments the A-domain sequences of engineered microcompartment proteins herein described comprise 3DKT: LLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGH YPLEKRVEECLRGGKIITTPRIEDALVVSER (SEQ ID NO: 64), GI 501179632: LREGTSNPKLALPSSASDYPAAIAAALNQLRLAGVNGPYAVVLGAGVYTALSGG DDEGYPVFRHIESLIDGKIVWAPAIEGGFVLSTR (SEQ ID NO: 65), GI 490598858: LLTEDGIVKFPISNWSEGENPFKDISIGLAKFIENGIVGRKALVVSPNLFVQLQRIQ PGTGTTEYDRINKLLDGNIFSTPVLKDDKAVLVCSE(SEQ ID NO: 66), GI 501923113 ILNAEGAQKLQISDWGQGENPYTDIVKAINMIREKGIVGRFVLCLSQSLYFDLQRI QQGTGMTEAQRISSMIGNLYNVPVIKGKKAALICAE (SEQ ID NO: 67), and GI 496662878: LLTVKGSSKIKKSDWSQGENSFADITAGVAQLAKTGYLGRYALVVSPDLFLDLQ RLQPNTGLLEIDRIKKLIGDNVYMTSVMGPGKAVLVCAE (SEQ ID NO: 68).

In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the P-domain which following sequence alignment provides a consensus sequence MDNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WG (SEQ ID NO:3) for the first fragment, the consensus sequence LPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK LARAEDRLIFN GYAEAGIEG (SEQ ID NO:5) for the second fragment and the consensus sequence GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT (SEQ ID NO:7) for the third fragment. In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise P-domain having a primary sequence with at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 3, 5- and 7.

An exemplary encapsulin shell protein from T. maritima has a SEQ ID NO: 47 shown in FIG. 5, comprising the P-domain, E-loop and A-domain.

In some embodiments, the encapsulin proteins herein used for constructing the engineered microcompartment proteins comprise the encapsulin proteins from the protein family PF04454 (Linocin_M18, Encapsulating protein for peroxidase), COG1659, and the DUF2184 superfamily. Proteins in this family are found in eubacteria and archaea, and can form nanocompartments within the bacterium which contain ferritin-like proteins or peroxidases, enzymes involved in oxidative-stress response. Detailed information about this protein family can be found in the pfam website as will be understood by a person skilled in the art (see the website http://pfam.xfam.org/family/PF04454 at the filing date of the present disclosure). A sequence alignment of 44 exemplary members from the PF04454 family are shown in FIG. 6.

In some embodiments, the encapsulin proteins herein used for constructing the engineered microcompartment proteins comprise the members of Phage capsid pfam05065 (see the website http://pfam.xfam.org/family/PF05065 at the filing date of the present disclosure) and HK97 family of viral capsid proteins (TIGRO1554) that have at least 22% identity or at least 40% similarity to SEQ ID NO:1.

In some embodiments, encapsulins herein described comprise homologous proteins of the encapsulin protein or SEQ ID NO: 1 or SEQ ID NO: 47 with at least 22% sequence identity or 40% sequence similarity, in which one or more residues forming the P-domain, E-loop and A-domain are replaced with a functionally equivalent residue.

A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical characteristics include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.

In embodiments according to the instant disclosure, a microcompartment protein is engineered to introduce in the encapsulin protein a target protein and at least one protease cleavage site configured to allow release of the target protein from the engineered microcompartment protein upon cleavage of the at least one protease cleavage site in the engineered microcompartment protein.

In particular in embodiments herein described:

a target protein having an N-terminus, a C-terminus and a sequence up to 80 amino acids in length is inserted at the N-terminus of the first segment of the P-domain of the encapsulin protein alone or together with a linker and/or a tag;

at least one first protease cleavage site is inserted between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein alone or together with a linker and/or a tag; and at least one second protease cleavage site is inserted, alone or together with a linker and/or a tag, within 9 to 17 amino acids and/or 0-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein to enable digestion of the encapsulin and release of the target protein.

As used herein, in relation to proteins, the term “insertion” of a first protein or fragment thereof in a second protein or fragment thereof refers to the introduction of the first protein or fragment thereof in between two adjacent amino acids of the first protein or fragment thereof. As a result, an inserted first protein is located in between the two adjacent amino acids of the second protein.

In particular, an insertion of a first protein in a second protein is performed by forming a first covalent bond between the N-terminal amino acid of the first protein with a first amino acid of the two adjacent amino acids the second protein, and a second covalent bond between the C-terminal amino acid of the first protein with a second amino acid of the two adjacent amino acids of the second protein. As would be understood by a skilled person, a covalent bond between two amino acids in a protein is typically a peptide bond, which is a covalent bond between a carboxyl group and an amino group of two molecules or portions thereof, which results in releasing a molecule of water.

Accordingly, an insertion of a second protein in a first protein when performed at a protein level typically results in breaking the peptide bond between the two adjacent amino acids of the first protein and forming two new peptide bonds: one between one of the two adjacent amino acids of the first protein and the N-terminal amino acid of the second protein and the other peptide bond formed between the other one of the two adjacent amino acid of the first protein and the C-terminal amino acid of the second protein.

In embodiments of the disclosure, insertion at the N-terminus of the first segment of the P-Domain is performed at the position between the N-terminus of the first segment of the P-Domain and the adjacent residue upstream in the encapsulin protein. For example, in embodiments where the encapsulin protein has SEQ ID NO: 1, the non-native protein can be introduced at an insertion position -1 relative to the N-terminus of the first segment of the P-Domain of the encapsulin protein. As a consequence, in embodiments where the encapsulin protein has SEQ ID NO: 1 the target protein or an insertion region comprising the target protein optionally together with a tag and/or a linker can be inserted between the adjacent residues X1 and X2 of the microcompartment protein. A schematic illustration of such insertion is illustrated in FIG. 7A. In some embodiments, the insertion region comprising the target protein alone or together with at least one tag and/or one linker comprises up to 80 amino acid residues.

In embodiments of the disclosure, insertion of the at least one first protease cleavage site between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein is performed at the position between the N-terminus of the first segment of the P-Domain and the adjacent residue upstream in the target protein. For example, in embodiments where the encapsulin protein has SEQ ID NO: 1, the target protein can be introduced at an insertion position −1 to position relative to the N-terminus of the first segment of the P-Domain of the encapsulin protein. As a consequence in embodiments where the encapsulin protein has SEQ ID NO: 1 the at least one first protease or an insertion region comprising the at least one first protease optionally together with a tag and/or a linker can be inserted between the residues X2 of the microcompartment protein and the residue immediately upstream residue X2. A schematic illustration of such insertion is illustrated in FIG. 7B in which the residues of the inserted target proteins are indicated as AA. In some embodiments, the insertion region comprising the at least one first protease alone or together with at least one tag and/or one linker, comprises up to 22 amino acid residues.

In embodiments of the disclosure, insertion of the at least one second protease cleavage site within 9-17 and/or 0-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein, can be performed at any one of the 9 to 17 amino acids and/or 0 to 8 adjacent residues upstream of the C-terminus of the E-Loop and/or 2-14 amino acids downstream of the N-terminus of the A-Domain.

As a consequence in embodiments where the encapsulin protein has SEQ ID NO: 1 the at least one second protease or an insertion region comprising the at least one second protease optionally together with a tag and/or a linker, can be inserted between any one of residues X57 and X74 of the microcompartment protein and/or between any one of residues X132 to X144 of the microcompartment protein. In particular, in SEQ ID NO: 1 residues X57 to X65 define the loop region of the E-loop domain (the first unstructured region of the E-Loop between beta-strands 132 and 133 of the E-Loop of the protein and residues X66 to X74 define beta strand β3 of the E-loop, and residues X132 to X144 define within the beta-strand β5 and the subsequent second unstructured region of the A Domain of the protein as will be understood by a skilled person.

A schematic illustration of such insertion is illustrated in FIG. 7C and FIG. 7D. In some embodiments, the insertion regions comprising the at least one second protease alone or together with at least one tag and/or one linker, can independently comprise up to 22 amino acid residues.

Exemplary sequences showing possible insertion points for the at least one second protease with bolded and bolded italics fonts are YSAVPLGRL EEIEGPAEGV

(SEQ ID NO. 4), and LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6).

Preferred insertion points for the at least one second protease cleavage site in SEQ ID NO: 1 are X₅₇ (corresponding to V57 in constructs exemplified in the example section of the present disclosure); X₆₀ (corresponding to D60 in constructs exemplified in the example section of the present disclosure); X₇₁ (corresponding to K71 in constructs exemplified in the example section of the present disclosure); and X₁₃₉ (corresponding to K138 in constructs exemplified in the example section of the present disclosure) which can be provided alone or in any combination selected in view of the resulting engineered microcompartment protein as will be understood by a skilled person upon reading of the present disclosure.

Exemplary sequences of engineered microcompartment proteins enclosing the above insertion points for the at least one second protease cleavage site are reported below wherein the related residues are reported in bold fonts.

(SEQ ID NO: 4) YSAVPLGRL E E IE G PAEGVQ AGVR Q VLP (SEQ ID NO: 6) LL NASGnL K LPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR

In embodiments herein described the engineered microcompartment proteins can be cage forming or non-cage forming depending on the positioning of the at least one second protease cleavage site.

The wording “cage forming” as used herein indicates an engineered microcompartment protein configured to form upon translation within cytoplasm of a cell or in a cell free environment, an encapsulin like microcompartment as described herein. Conversely, the wording non-cage forming as used herein indicates an engineered microcompartment protein configured not to form upon translation within a cytoplasm of a cell or in a cell free environment, an encapsulin like microcompartment as described herein

In particular in some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site can be within the loop region of the E-loop domain (the first unstructured region of the E-Loop, between beta-strands β2 and β3 of the protein) to provide a cage forming engineered microcompartment protein. Examples are insertions at any of positions X57 and X65 in SEQ ID NO 1, and any of the position 11 (E) to 19 (V) (EIEGPAEGV- SEQ ID NO: 139) in SEQ ID NO; 4. Representative examples are provided by insertions at residues V57 and D60 in T. maritima encapsulin (see Examples 17 to 24). In particular, in order to obtain cage forming microcompartment proteins one insertion can be performed at any one of the residues (see e.g. the insertion of a GG-ENLYFQG-GG SEQ ID NO: 140 or residue of a same or smaller dimension after 1 residue in the region of the Enc from T. maritima which already has 9 amino acids).

In some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site on the β5 beta strand prior to α5 of the A-Domain to provide a cage forming engineered microcompartment protein. Examples are insertions at any of positions X132 and X144 in SEQ ID NO 1, and any of the position 2 (L) to 14 (A) of sequence LNASGnLKLPLSA (SEQ ID NO: 141) in SEQ ID NO: 6.

In some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site in the loop region of the E-loop (in the first unstructured region of the E-loop between β2 and β3) and in the A domain and the resulting engineered microcompartment proteins are also expected to be cage forming.

In some embodiments, an engineered microcompartment protein can be designed to include at least one second protease cleavage site in the E-loop domain within the beta-strand β3 to provide a non-cage forming engineered microcompartment protein. In particular, a disruption of cage-formation occurs if there is an insertion within β3 of the E-loop (e.g. X71 in SEQ ID NO; 1 and K71 construct in the examples) while cage formation would not be disrupted if there is an insertion after X139 in SEQ ID NO; 1 (see K138 constructs in the examples). In some of these embodiments, the addition of this site can improve the kinetics of peptide release. An example is an insertion at any of positions X66 and X74 in SEQ ID NO 1, and any of the position 20 (Q) to 28 (P) (Q AGVRQVLP SEQ ID NO: 142) in SEQ ID NO; 4. A representative example is provided by insertion following residue K71 in T. maritima encapsulin. (see Examples 17 to 24).

Non-cage forming insertion point for the at least one second protease are expected to be dominant with respect to the cage formation of engineered microcompartment proteins of the present disclosure.

Accordingly, engineered microcompartment proteins comprising the at least one protease cleavage site in the β3 strand of the E Loop are expected to be non cage forming when in combination with additional second protease cleavage sites in the A Domain are and/or other regions of the E-Loop also expected to be non cage forming. In particular, any insertion within β3 strand of the E-loop domain is expected to be non-cage forming. As a consequence engineered microcompartment proteins comprising the at least one protease cleavage site in the β3 strand of the E Loop and in the loop region of the E-loop (in the first unstructured region of the E-loop between β2 and β3) are also expected to be non cage forming.

In some embodiments the at least one first protease cleavage site and the at least one second protease cleavage site comprise a same protease cleavage sites. In some embodiments the at least one first protease cleavage site and the at least one second protease cleavage site comprise different protease cleavage sites.

The wording “protease cleavage site in the sense of the disclosure indicates target sites for proteolytic cleavage by enzymes such peptidases, proteases or proteolytic cleavage enzymes which break peptide bond between amino acids in proteins. The general nomenclature of cleavage site positions of the substrate were formulated by Schechter and Berger, 1967 [9]and Schechter and Berger, 1968 [10] Accordingly, the cleavage site is designated between P1-P1′, incrementing the numbering in the N-terminal direction of the cleaved peptide bond (P2, P3, P4, etc. . . . ). On the carboxyl side of the cleavage site the numbering is incremented in the same way (P1′, P2′, P3′ etc.).

Protease cleavage sites that can be inserted in engineered microcompartment proteins of the disclosure comprise regions up to 25 residues. In particular, protease cleavage sites are inserted in a configuration which makes them surface accessible. In some embodiments protease cleavage site are included in an unstructured segment or within an alpha helical or beta sheet secondary structured segment. Exemplary protease cleavage sites that can be inserted in engineered microcompartment proteins herein described comprise TEV protease cleavage sites with sequence ENLYFQG, (SEQ ID NO:69) which is unstructured and others identifiable by a skilled person upon reading of the present disclosure (see also Table 2 and Example 3).

In some embodiments of the engineered microcompartment protein herein described, the at least one cleavage site is comprised within an inserted region of up to 25 residues further comprising linkers and/or tags as will be understood by a skilled person upon reading of the present disclosure.

In embodiments herein described target proteins that can be inserted comprise any protein having 1 to 80 residues possibly comprised within an inserted region of up to 80 residues further comprising linkers and/or tags as will be understood by a skilled person upon reading of the present disclosure.

The term “protein” as used herein indicates a polypeptide with secondary, tertiary, and possibly quaternary structure. The protein's secondary, tertiary, and quaternary structure can occur on a variety of length scales (tenths of A to nm) and time scales (ns to s), so that in various instances the secondary, tertiary and possibly quaternary structures are dynamic and not perfectly rigid.

The term “polypeptide” as used herein indicates a polymer composed of two or more amino acid monomers and/or analogs thereof wherein the portion formed by the alpha carbon, the amine group and the carboxyl group of the amino acids in the polymer forms the backbone of the polymer. As used herein the term “amino acid”, “amino acid monomer”, or “amino acid residue” refers to any of the naturally occurring amino acids, any non-naturally occurring amino acids, and any artificial amino acids, including both D and L optical isomers of all amino acid subsets. In particular, amino acid refers to organic compounds composed of amine (—NH2) and carboxylic acid (—COOH), and a side-chain specific to each amino acid connected to an alpha carbon. Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity, and pKa. Amino acids can be covalently linked to forma polymer through peptide bonds by reactions between the amine group of a first amino acid and the carboxylic acid group of a second amino acid.

The term “polypeptide” includes amino acid polymers of any length including full length proteins, as well as analogs and fragments thereof. The polypeptide provides the primary structure of a protein wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of amino acid residues covalently linked by peptide bond.

In particular, in some embodiments of the present disclosure, the target protein of the engineered microcompartment protein of the disclosure can be a protein which is non-native to the bacterial cell where the engineered microcompartment protein is configured to be expressed based on the experimental design.

The wording “native” as used herein with reference to a compound and a cell, identifies a compound, molecule or structure naturally provided and in particular produced in the cell. Therefore, a native protein or a native substrate when described in connection with a cell, refers to a protein and/or substrate that is itself naturally provided and in particular, produced in the referenced cell. Conversely, the term “non-native” as used herein with reference to a protein and/or a substrate in connection with a cell, refers to a protein and/or substrate that is itself not naturally produced in the referenced cell.

In embodiments wherein the target protein is a non-native protein, the non-native protein herein described include toxic non-native proteins and non-toxic non-native proteins which in some cases can be degraded within a target bacterial cell where expression production and/or purification of the non-native protein is desired. Accordingly, engineered microcompartment proteins compositions, in some embodiments are configured to allow compartmentalization of certain toxic or non-toxic proteins in cells where said proteins are non-native, thus shielding the cell from toxicity from said toxic non-native proteins or shielding the non-native proteins from the cell environment.

Exemplary non-toxic non-native proteins that can be introduced as target protein in the engineered microcompartment proteins of the disclosure include proteins that can be proteolyzed by protease within the host cell and in in particular, proteins which are particularly sensitive to native proteases present in the host cells. In such cases, the engineered microcompartment proteins can protein the non-native proteins from proteolysis in the host. Exemplary non-native non-toxic proteins include proteins susceptible to Lon protease, OmpT and ClpXP in E. coli. Non-toxic non-native proteins also include proteins that are difficult to fold, including those that require disulfide bonds for proper folding and function. The engineered microcompartment proteins can provide an enclosed structure to prevent aggregation and facilitate proper folding.

The term “toxic non-native protein”, as used throughout, refers to a protein or peptide that is itself not naturally produced by a reference cell and is toxic to the host cell when provided or produced in said cell.

In particular, toxic non-native proteins in the sense of the disclosure in connection with cells where they are expressed produced and/or purified indicates proteins or peptides that are not native to said cell and can react with a native cellular target substrate to provide cell damage by triggering a series of linked biological or chemical reactions within the cell resulting in damage to said cell.

The wording “native cellular target substrate” or “native cellular substrate” as used herein indicates a compound molecule or structure that is naturally occurring in a cell and is a part of reactions taking place in the cell to keep the cell alive. Exemplary native cellular target substrates in the sense of the disclosure comprise native cellular lipids, proteins, nucleic acids and/or related cellular structures, such as cell membrane or cell chromatin.

Exemplary reactions between a native cellular target substrate and a non-native protein, particularly a toxic non-native protein, which exemplary reactions trigger a series of linked biological or chemical reactions in the cell resulting in a damage to the cell, comprise binding and/or bond cleavage resulting in disruption and/or inactivation of the cellular target substrate. For example, targeting of membrane lipids damages the cell membrane which, on its turn, impacts the state of cell electrolytes, e.g. calcium, which when constantly increased, induces apoptosis.

The word “damage” as used herein refers to a physical harm caused to a cell in such a way as to impair its normal function. In particular, cell damage can occur as a result of disruption the normal homeostasis of an affected cell. Among other causes, cell damage can be due to physical, chemical, or, biological, factors resulting from targeting of cell components such as DNA and the cell membrane. Cell damage can be reversible or irreversible. Depending on the extent of injury, the cellular response may be adaptive and where possible, homeostasis is restored. Cell death occurs when the severity of the injury exceeds the cell's ability to repair itself and can occur by necrosis or apoptosis.

Toxicity in the sense of the disclosure in particular occurs when a non-native protein interferes with the normal proliferation and homeostasis of the microorganism and the visible result is slower growth rate, low final cell density, and death ([11]-[12]) Toxicity of a non-native protein can therefore be detected with reference cell growth before production of a non-native toxic protein (basal growth) and after detection of possible toxicity of vectors or other expression system for production of the non-native protein within a cell which can be performed with approaches discussed for example in reference (2) or otherwise identifiable by a skilled person upon reading of the present disclosure. After control of basal growth and of toxicity of the expression system, the culture can be grown until the expression of the non-native protein. Following expression of the non-native protein, if the non-native protein is toxic, cell growth will be impaired or arrested depending on the level of toxicity. In some cases, the level of toxicity of a non-native protein can be dependent on a threshold of host tolerance. In such situations, toxicity of a non-native protein can be dependent on the level of expression of the non-native protein in comparison with the threshold of host tolerance which should be reached and exceeded for the protein to have toxicity as will be understood by a skilled person.

Examples of proteins or peptides that are toxic and therefore harmful to a cell include antimicrobial peptides, as well as proteases and lysins, which are harmful to bacterial cells through direct targeting of cytoplasmic, membrane, DNA or protein synthesis.

In particular, toxic non-native protein that can be included in engineered microcompartment proteins of the instant disclosure, are toxic proteins or peptides that are non-native to the cell where they are produced and that have a native cellular target substrate which is a native membrane substrate.

The wording “membrane” as used herein indicates a biological membrane that separates the interior of a cell from the outside environment and can have different structure and configurations in different type of cells as will be understood by a skilled person. In particular, the wording “membrane” as used herein is intended to encompass: i) a cell plasma membrane (also identified as inner membrane in Gram negative bacteria) typically formed by a phospholipid bilayer with embedded proteins, ii) the outer membrane of Gram-negative bacteria formed by a phospholipid bilayer with embedded proteins different in composition from the inner membrane (e.g. rich in lipopolysaccharide), as well as iii) the cell wall, a structural layer that surrounds some types of cells, situated outside the cell membrane and is mainly composed of peptidoglycan (amino acids and sugars). In particular, cell wall can be made of peptidoglycan (also called murein), which is made from polysaccharide chains cross-linked by unusual peptides containing D-amino acids.

The wording “native membrane substrate” as used herein indicates a compound that is naturally located in the membrane of a cell in the sense of the disclosure and in particular in the membrane of the cell where the toxic non-native protein is expressed or to be expressed. Native membrane substrates comprise proteins, peptidoglycans, and lipids located in the plasma membrane, inner membrane, outer membrane or cell wall of a cell in the sense of the disclosure.

An exemplary native membrane substrate that can be targeted by toxic proteins herein described are peptidoglycan and lipopolysaccharide (LPS) biosynthesis proteins, which are enzymes such as MraY, LpxK, KdtA, LpxL, LpxM, MraG, FtsW catalyzing biosynthesis of peptidoglycans of the cell wall and LPS in the outer membrane. In particular MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate [13]) Non-native toxic proteins such as LysE react with peptidoglycan with resulting damage to the cell wall and to the cell. LpxK is a gene encoding tetraacyldisaccharide 4′-kinase, an enzyme that phosphorylates the 4′-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipopolysaccharide lipid A. This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. KdtA is a gene encoding 3-deoxy-D-manno-octulosonic acid transferase, which is involved in lipopolysaccharide (LPS) biosynthesis. This enzymes catalyzes the transfer of two 3-deoxy-D-manno-octulosonate (Kdo) residues from CMP-Kdo to lipid IV(A), the tetraacyldisaccharide-1,4′-bisphosphate precursor of lipid A. LpxM is a gene encoding Lipid A biosynthesis myristoyltransferase, an enzyme that catalyzes the transfer of myristate from myristoyl-acyl carrier protein (ACP) to Kdo(2)-(lauroyl)-lipid IV(A) to form Kdo(2)-lipid A. In vitro, the protein can acylate Kdo(2)-lipid IV(A), but the acylation of (Kdo)2-(lauroyl)-lipid IV(A) is about 100 times faster. In vitro, the protein can use lauroyl-ACP but displays a slight kinetic preference for myristoyl-ACP. LpxL is a gene encoding Lipid A biosynthesis lauroyltransferase, an enzyme that catalyzes the transfer of laurate from lauroyl-acyl carrier protein (ACP) to Kdo(2)-lipid IV(A) to form Kdo(2)-(lauroyl)-lipid IV(A). This enzyme has 10-fold selectivity for lauroyl-ACP over myristoyl-ACP. In vitro, this enzyme can also catalyze a slow second acylation reaction leading to the formation of Kdo(2)-(dilauroyl)-lipid IV(A). FtsW is a gene encoding lipid II flippase FtsW protein, a cell division protein that transports lipid-linked peptidoglycan precursors from the inner to the outer leaflet of the cytoplasmic membrane. This protein is required for localization of Ftsl and may also play a role in the stabilization of the FtsZ ring during cell division.

Additional, native membrane substrates that can be targeted by native toxic proteins herein described are peptidoglycans comprising a pentapeptide motif A(D/N)LXX (SEQ ID NO:8), where X can be any amino acid with the central position in the pentapeptide motif (also designated as position i) being usually a leucine, position i−2 being usually an alanine and the two subsequent positions (i+1 and i+2) configured so that the side chains of positions i−2 and i point into the hydrophobic interior of the protein while the side chains of positions i−1, i+1 and i+2 are exposed on the surface of the proteins. Those peptidoglycans can be targeted for example by non-native lysin proteins with a peptidase domain which can be identified for example using a BLAST search on NCBI. For instance, Ply500 has a pfam02557: VanY: D-alanyl-D-alanine carboxypeptidase motif which would react with a pentapeptide motif in a peptidoglycan. Additional lysins can be identified by a skilled person upon reading of the present disclosure.

Further native membrane substrates that can be targeted by native toxic proteins are peptidoglycans comprising a sugar motif, such as GlcNAc-X-GlcNAc with X being any amino acid and other sugar motifs identifiable by a skilled person. These native membrane substrates can be targeted by non-native lysins proteins having an amidase domain, which can also be identified for example using a BLAST search, and additional lysins identifiable by a skilled person.

Additional native membrane substrates that can be targeted by native toxic proteins herein described are phospholipids in the inner membrane. In those embodiments, one or more non-native toxic proteins can bind to lipid and inhibit proper structure of the lipid bilayer membrane, causing holes to form in the membrane. Examples of toxic proteins targeting phospholipids are AMPs having alpha helical or beta-sheet that disrupt inner membrane such as cecropin, magainin, melittin, and protegrin I.

Further native membrane substrates that can be targeted by native toxic proteins are lipids in the outer membrane (e.g., Lipid II and LPS). Examples of toxic proteins targeting lipids of the outer membrane comprise cationic antimicrobial peptides such as cecropin P1, defensins, and nisins.

Additional native membrane substrates that can be targeted by native toxic proteins herein described are outer membrane proteins such as integral outer membrane proteins folding into antiparallel beta-barrels. (e.g. proteins belonging to the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA). Examples of toxic proteins targeting lipids of the outer membrane proteins are cationic antimicrobial peptides. An example is inhibition of OmpF porin by HP(2-20) peptide. Additional cationic antimicrobial peptides expected to be found in ([14] [15]).

In embodiments herein described, the non-native proteins are expressed in constructs where one or more non-native proteins is fused to at least one encapsulin protein herein described to form protein to provide an engineered microcompartment protein, in which the non-native protein can be later released by cleaving from the engineered microcompartment protein. Some non-native proteins have an extended, non-helical structures (e.g., LL-37, Apidaecin Ia) while others have an alpha helical structure (e.g., HBCM2—which is a hybrid of cecropin and melittin, which are both alpha helical).

In some embodiments, “toxic non-native protein” that can be used as target protein in engineered microcompartment protein and in related cells compositions methods and systems of the instant disclosure comprise antimicrobial peptides targeting cell membrane, proteases targeting proteins in a native cell membrane as defined herein, and lysins as will be understood by a skilled person.

The term “Antimicrobial peptides” or “AMPs”, indicates peptides generally less than 200 amino acids and typically between 12 and 50 amino acids, having two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (generally >50%) of hydrophobic residues and having an antimicrobial activity as would be understood by a skilled person. The secondary structures of AMPs typically follow 4 themes, including i) a-helical, ii) β-stranded due to the presence of 2 or more disulfide bonds, iii) β-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain, and iv) extended as would be understood by a skilled person. The final cellular configuration of AMPs typically contains hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. AMPs can cause cell damage by reacting with membrane components to induce membrane permeabilization or with a range of cytoplasmic targets. In some instances, amino acid composition, amphipathicity, cationic charge and size of AMPs allow them to attach to and insert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’ or ‘toroidal-pore’ mechanisms. In some instances, AMPs can bind target intracellular molecules which are crucial to cell viability thus resulting in cell damage through inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of DNA, RNA, and protein synthesis, and/or inhibition of enzymes identifiable by a skilled person. In general, the antimicrobial activity of these peptides is determined by measuring the minimal inhibitory concentration (MIC), which is the lowest concentration of drug that inhibits bacterial growth. Antimicrobial peptides have been demonstrated to kill Gram negative and Gram-positive bacteria, enveloped viruses, fungi and even transformed or cancerous cells. In some embodiments, the AMPs herein used for the constructing engineered microcompartment proteins have an extended or alpha helical structure.

In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise cationic AMPs that target phospholipids in the inner membrane, such as cecropin, magainin, melittin, and protegrin I, or derivatives thereof.

In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise cationic AMPs that target native outer membrane proteins, such as HP(2-20) peptide capable of targeting and inhibiting OmpF porin as well as SMAP-29 and CAP-18 both capable of targeting and inhibiting outer membrane protein I (Oprl).

In particular, the term “cecropins” indicate AMPs of about 31-37 amino acid residues having alpha helical conformation and being capable of targeting native membrane substrates of both Gram-positive and Gram-negative bacteria. Cecropins isolated from insects other than Hyalophora cecropia (Cecropia moth) are also known as bactericidin, lepidopterin, sarcotoxin, and additional names identifiable by a skilled person. Exemplary cecropin comprise Cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK SEQ ID NO: 20) having a secondary structure with two a helices, and being capable of forming a ion channels at low peptide to lipid ratios and pores at high peptide to lipid ratios as will be understood by a skilled person. Exemplary cecropins also comprise: Cecropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL SEQ ID NO: 21) having two a helices in the secondary structure, CECD from Aedes aegypti (Yellowfever mosquito), Papiliocin (A lepidopteran) from Papilio xuthus an Asian swallowtail butterfly, and Cecropin P1, an antibacterial peptide from Ascaris suum, a parasitic nematode that resides in the pig intestine. Cecropin derivatives comprise peptides modified cecropins (e.g. cecropin A, and cecropin B). In some embodiments, derivatives of cecropins have anticancer properties and are called anticancer peptides (ACPs) ([16] In particular hybrid ACPs based on Cecropin A have been studied for anticancer properties ([17])

The term “magainins” indicate a class of antimicrobial peptides found in the African clawed frog Xenopus laevis identifiable by a skilled person

The term “melittin” indicates the principal active component of apitoxin (bee venom), a powerful stimulator of phospholipase A2 as will be understood by a skilled person. Melittin is a peptide consisting of 26 amino acids with the sequence GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:22).

The term “protegrins” indicates small peptides containing 16-18 amino acid residues. The amino acid composition of protegrins contains six positively charged arginine residues and four cysteine residues. Their secondary structure is classified as cysteine-rich β-sheet antimicrobial peptides, AMPS that display limited sequence similarity to certain defensins and tachyplesins. In solution, the peptides fold to form an anti-parallel β-strand with the structure stabilized by two cysteine bridges formed among the four cysteine residues. Protegrins bind to lipopolysaccharide, a property that may help them to insert into the membranes of gram-negative bacteria and permeabilize them. The term “defensins” as used herein identifies small cysteine-rich cationic proteins found in vertebrates, invertebrates and plants. Defensins have 18-45 amino acids including six to eight conserved cysteine residues. Most defensins function by binding to the microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients.

The term “nisins” as used herein identifies a polycyclic peptide produced by the bacterium Lactococcus lactis having 34 amino acid residues, including the uncommon amino acids lanthionine (Lan), methyllanthionine (MeLan), didehydroalanine (Dha), and didehydroaminobutyric acid (Dhb). These unusual amino acids are provided by posttranslational modification of the precursor peptide. In these reactions, a ribosomally synthesized 57-mer is converted to the final peptide. The unusual amino acids originate from serine and threonine, and the enzyme-catalyzed addition of cysteine residues to the didehydro amino acids result in the multiple (five) thioether bridges.

In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise HBCM2, HBCM3 and Apidaecin Ia.

The term “HBCM2” and “HBCM3” refers to hybrid (HB) of silk moth cercropin (C) and bee melittin (M) antimicrobial peptides. These are residue optimized peptides that have high efficacy against Pseudomonas aeruginosa [18, 19]. They originate from cercropin from silk moth and melittin from bees, which are both alpha helical in secondary structure and therefore, HBCM2 and HBCM3 are also thought to be alpha helical in structure.

The term “Apidaecin Ia” (AP) refers to a series of small, proline-rich, 18- to 20-residue peptides produced by insects. They are the largest group of Pro-rich antimicrobial peptides known to date. Structurally, apidaecins consist of two regions, the conserved region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many Gram-negative bacteria by special antibacterial mechanisms. The mechanism of action by which apidaecins kill bacteria involves an initial non-specific binding of the peptides to an outer membrane (OM) component. This binding is followed by invasion of the periplasmic space, and by a specific and essentially irreversible combination with a receptor/docking molecule that may be a component of a permease-type transporter system on inner membrane (IM). In the final step, the peptide is translocated into the interior of the cell where it meets its ultimate target. Evidence that apidaecins are non-toxic for human and animal cells is a prerequisite for using them as novel antibiotic drugs.

The term “protease” (also called a peptidase or proteinase or proteolytic enzyme) indicates any enzyme that performs proteolysis, (begins protein catabolism) by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. Proteases can be classified into seven broad groups based on the amino acid at the (protease's) active site used to perform a nucleophilic attack on the substrate: Serine proteases—using a serine alcohol; Cysteine proteases—using a cysteine thiol; Threonine proteases—using a threonine secondary alcohol; Aspartic proteases—using an aspartate carboxylic acid; Glutamic proteases—using a glutamate carboxylic acid; Metalloproteases—using a metal, usually zinc; Asparagine peptide lyases—using an asparagine to perform an elimination reaction (not requiring water), as would be understood by a skilled person. In particular, Aspartic, glutamic and metallo- proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it. Serine, threonine and cysteine proteases use a nucleophilic residue in attack (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme. Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, and elastase). Some proteases can be promiscuous and react with wide range of protein substrates. This is the case for example of digestive enzymes such as trypsin which have to be able to cleave the array of proteins ingested into smaller peptide fragments. Promiscuous proteases typically bind to a single amino acid on the substrate and so only have specificity for that residue. For example, trypsin is specific for the sequences . . . K\ . . . or . . . R\ . . . (‘\’=cleavage site). Some proteases are specific and only cleave substrates with a certain sequence or amino acid structure. Proteases, being themselves proteins, can be cleaved by other protease molecules, sometimes of the same variety. This acts as a method of regulation of protease activity. Some proteases are less active after autolysis (e.g. TEV protease) whilst others are more active (e.g. trypsinogen). Specific proteases targeting native membrane substrates are expected to be usable in methods and systems described herein.

In some embodiments herein described, the proteases inserted as target protein in engineered microcompartment proteins herein described are generally non-specific in their recognition site, meaning their recognition sequence are recognized by a large number of protein substrates. A lack specificity of a protease can be due to a short recognition sequence and/or promiscuity of the protease. Proteases can also be used for cleaving toxic non-native proteins from the engineered microcompartment proteins. Such proteases used for cleaving toxic non-native proteins would be highly specific for a unique recognition sequence that is not commonly found in protein substrates. For instance, TEV protease has a relatively long recognition sequence (ENLYFQ\S(orG)) (SEQ ID NO: 70) that is not commonly found in other proteins. Therefore, it can be used to specifically digest the engineered encapsulin microcompartment and/or release the toxic protein from the encapsulin microcompartment to obtain the toxic protein of interest without resulting in non-specific side products and damage to host cells which in some instance can cause death to the cell. In the case of cellular expression, these specific proteases can digest the encapsulin microcompartments with limited digestion of other cellular proteins that may result in cellular toxicity.

In particular, specific proteases that target membrane substrates can be compartmentalized with methods herein described. Exemplary specific proteases comprise intramembrane proteases that cleave the transmembrane domain of proteins, such as YaeL from E. coli and SpoIVFB from Bacillus subtilis, additional proteases described in ([20]). In particular, intramembrane proteases such as YaeL (also called RseP) in Escherichia coli play a role in coordinating cell growth and cell division through intramembrane proteolysis of RseA. SpoIVFB is an intramembrane metalloprotease, in Bacillus subtilis that cleaves factors required for sporulation (processing of pro-sigma-K to active SigK). Additional proteases such as endopeptidases that target peptidoglycan. The term “endopeptidases” identifies proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. The relevant peptidase domain can be found by BLAST search on NCBI as will be understood by a skilled person. Additional proteases that target membrane substrates can be identified by a skilled person upon reading of this disclosure.

The term “lysins”, also known as endolysins or murein hydrolases, indicates hydrolytic enzymes produced by bacteriophages in order to cleave the host's cell wall during the final stage of the lytic cycle or natively by bacteria themselves in order to remodel their own cell wall. Usually lysins are monomeric proteins with a 25 to 40 kDa range in size. A notable exception is the streptococcal PlyC endolysin, which is 114 kDa and composed of two different gene products, PlyCA and PlyCB, with a ratio of eight PlyCB subunits for each PlyCA in its active conformation as will be understood by a skilled person. Lysins comprise an at least one domain catalyzing the hydrolysis of peptidoglycan and a domain binding to the cell wall substrate. In lysins, the catalytic domain is responsible for the cleavage of peptidoglycan bonds, and can be one of the following five types of lysin catalytic domain: Endo-P-N-acetylglucosaminidase, N-acetylmuramidase (lysozyme-like), Endopeptidase, N-acetylmuramoyl-L-alanine amidase, γ-D-glutaminyl-L-lysine endopeptidase identifiable by a skilled person. In lysins, the cell-binding domain (CBD) binds to a specific substrate found in the host bacterium's cell wall, usually a carbohydrate. In contrast to the catalytic domain, the cell-binding domain is variable, which allows a great specificity and decreases bacterial resistance. Binding affinity to the cell wall substrate tends to be high, possibly so as to sequester onto cell wall fragments any free enzyme, which could compete with phage progeny from infecting adjacent host bacteria. In lysins usually, two or more different catalytic domains are linked to a single cell-binding domain. This is typical in many staphylococcal lysins as well as the streptococcal PlyC holoenzyme, which contains two catalytic domains. Catalytic domains are highly conserved in phage lysins of the same class. In monomeric lysins, the catalytic domain is typically at the N-terminal end of the protein and the cell binding domain is located at the C-terminal end of the protein and the two domains are separated by a short linker region. Target cellular substrate of lysins are peptidoglycans, which consists of cross-linked amino acids and sugars which form alternating amino sugars: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Endo-β-N-acetylglucosaminidase lysins cleave NAGs while N-acetylmuramidase lysins (lysozyme-like lysins) cleave NAMs. Endopeptidase lysins cleave any of the peptide bonds between amino acids, whereas N-acetylmuramoyl-1-alanine amidase lysins (or simply amidase lysins) hydrolyze the amide bond between the sugar and the amino acid moieties. Finally, the recently discovered γ-d-glutaminyl-1-lysine endopeptidase lysins cleave the gamma bond between D-glutamine and L-lysine residues. Lysins typically target one of the five bonds in peptidoglycan (murein), the main component of bacterial cell walls, which allows the release of progeny virions from the lysed cell in the case of phage lysins and the remodeling of cell wall in the case of native bacterial lysins. These enzymes are being used as antibacterial agents due to their high effectiveness and specificity in comparison with antibiotics, which are susceptible to bacterial resistance.

In some embodiments, lysins that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise lysozyme-like lysins, such as Cpl-1 and Cpl-7 that target S. pneumoniae peptidoglycan, amidase lysins, such as PlyPSA that targets L. monocytogenes peptidoglycan and endopeptidases that target the pentapeptide motif of peptidoglycan, such as Ply500 that targets L. monocytogenes peptidoglycan and additional lysins described in reference ([21]).

In particular, the term “lysozyme like lysins” indicates lysins with a catalytic N-acetylmuramidase (lysozyme-like) domain, the term “amidase lysins” identifies with an amidase domain such as amidase 3 domain as shown in the website ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=119407 at the date of filing of the instant disclosure.

In some embodiments herein described, toxic non-native proteins are non-native proteins or peptides that act within the membrane either by direct interaction/disruption of the membrane or through inhibition of membrane biosynthesis proteins. Examples of such toxic non-native proteins include LysE protein from phiX174 bacteriophage and antimicrobial peptides [22]. LysE protein binds to and inhibits the peptidoglycan biosynthesis protein MraY located in the bacterial membrane, thus resulting in cell lysis, and antimicrobial peptides targeting the bacterial cell membrane, and/or targeting other cellular target substrate other than DNA [23].

In some embodiments herein described, target proteins introduced in engineered microcompartment protein are non-native AMPs lacking disulfide bonds, such as cecropin, melittin, and apidaecin AMPs.

In some embodiments described, the toxic non-native proteins introduced as target proteins of engineered microcompartment protein are pro-rich antimicrobial peptides. Examples of toxic non-native proteins that can be used in engineered microcompartment proteins, engineered microcompartment, related vectors, cells compositions methods and systems of the disclosure include apidaecin and antimicrobial peptides which target membrane substrates, and are harmful to bacterial cells (see Example 1).

In embodiments according to the instant disclosure a microcompartment protein is engineered to introduce at least one non-native protein having a sequence up to 80 amino acids in length at the N-terminus of the first segment of the P-domain of the microcompartment protein, together with at least one protease cleavage site inserted between the C-terminus of the non-native protein and the N-terminus of the first segment of the P-Domain of the microcompartment protein.

FIG. 8 shows another exemplary engineered microcompartment protein (SEQ ID NO: 23) comprising Apidaecin Ia peptide fused to the N-terminus of an encapsulin protein from M xanthus through a TEV protease cleavage site and a linker region.

In some embodiments, the target protein can be fused to the N-terminus of the encapsulin protein or encapsulin-like proteins. For example, the target protein can be inserted in an insertion region between the first and second position (the M in X1 and E in X2) of the encapsulin shell protein having the SEQ ID NO: 47 (see Example 12-15). In some of those embodiments, a linker can be comprised within the insertion region placed between the non-native protein and the N-terminus of the encapsulin protein. In some embodiments, the linker comprises a protease cleavage site in order to enable later cleavage of the target protein from encapsulin via a protease that specifically targets at the protease cleavage site. Exemplary recognition sequences and cleavage sites of proteases include the ones shown in Table 2 as well as others identifiable by a person skilled in the art.

Exemplary engineered microcompartments designed to include a peptide fused to the N-terminus of an engineered encapsulin protein through a protease cleavage site and a linker region are described in Example 17 of the present disclosure.

In some embodiments, the engineered protein is configured so that cleavage of the target protein from encapsulin via a protease that specifically targets at the protease cleavage site results in a target protein having N-terminal residues of the cleavage site attached to its C-terminus. In some of those embodiments the N-terminal residues of the cleavage site can be undesired as for example they can interfere with the activity of the cleaved target protein.

In those embodiments, the engineered microcompartment protein can be configured to include a proline residue between the N-terminus of the protease cleavage site and the C-terminus of the target protein. In those embodiments the N-terminal residues of the cleavage site attached to the target protein can be digested with carboxypeptidase that proteolyzes from the C-terminus of the target protein but does not have peptidase activity at proline residues (see Example 27).

Exemplary embodiments wherein insertion of a proline residue between the N-terminus of the protease cleavage site and the C-terminus of the target protein can be desired are provided by constructs including protease cleavage sites with the overall charge of their N-terminal residues interferes with the proper folding and/or activity of the target protein. Examples of these protease cleavage sites include: the enterokinase protease cleavage site (DDDDK SEQ ID NO: 71), the TEV protease cleavage site (ENLYFQ/G SEQ ID NO: 72), and the HRV-3C protease cleavage site (LEVLFQ/GP SEQ ID NO: 73) whose N-terminal residues have an overall negative charge, as well as the thrombin protease cleavage site (LVPR/GS SEQ ID NO: 74) whose N-terminal residues have an overall positive charge. These N-terminal residues may interfere with a target peptide depending on the configuration of the target peptide.

In those embodiments, a proline can be inserted between the N-terminal residue of the protease cleavage site (e.g., N-terminal D residue of the enterokinase protease cleavage site) and the C terminus of the target protein or a protease cleavage site with an overall net neutral charge can be selected in order to retain or improve the activity of the target peptide in its native state. Examples of such protease cleavage site include the recognition sequence for the Factor Xa IEGR (Table 2)

In general in embodiments, wherein a target peptide has a configuration which is known or expected to be incompatible with one or more protease cleavage sites (e.g. because of negatively or positively charged N terminal residues of the protease cleavage site or other incompatibilities), replacement of the protease cleavage site with an alternative protease cleavage which does not interfere with the target protein of interest can be performed. In the alternative placement of a proline in the construct between the N-terminus of the cleavage site and the C terminus of the target protein, can also be performed to allow digestion of the N-terminal residues of the cleavage site attached to the C-terminus of the target protein.

Protease cleavage sites can be tested to determine if following cleavage, the residual protease cleavage site on the C-terminus of the target protein interferes with the target protein activity by comparing the activity of the target protein with and without the residual protease cleavage site at its C-terminus. The target protein with and without the residual protease cleavage site at its C-terminus can be obtained by chemical synthesis methods (e.g., solid phase peptide synthesis) via commercial sources (e.g., Elim biopharmaceuticals). Activity of the target protein can be determined by an appropriate enzymatic or cell inhibition assay.

As an alternative, the target protein fused to a proline residue followed by the protease cleavage site followed by the engineered microcompartment protein can be translated in the cytoplasm of a cell and purified using methods identifiable by a skilled person. The purified material can be digested with the appropriate protease to obtain the target protein with the residual protease cleavage site on its C-terminus. The target protein with the residual protease cleavage site can be further digested with carboxypeptidase to obtain the target protein with a residual proline at its C-terminus. The activities of the target protein with the residual protease cleavage site versus the residual proline can be compared to each other as well as a chemically synthesized target protein with no residual amino acids at its C-terminus. This method will determine if any residual amino acids at the C-terminus of a target protein affects its activity.

In some embodiments, one or more protease sites can also be inserted within the encapsulin shell protein to enable full digestion of the encapsulin cage and thus full release of the non-native protein. The protease cleavage sites can be inserted within 1-8 amino acids adjacent to the C-terminus of the E-loop of the SEQ ID NO:1 at the β3 β-sheet region close to the P-domain. In some other embodiments, the protease cleavage sites can be inserted within 1-8 amino acids adjacent to the N-terminus of the A-domain at the surface-exposed region (see Examples 12-17).

In some embodiments, the engineered microcompartment protein can further include one or more tags inserted in the engineered microcompartment protein.

The term “tag” as used herein means protein tags comprising peptide sequences introduced onto a recombinant protein. Tags can be removable by chemical agents or by enzymatic means, such as proteolysis or splicing. Tags can be attached to proteins for various purposes: Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. These include chitin binding protein (CBP), and the poly(His) tag. The poly(His) tag is a widely-used protein tag; it binds to metal matrices. Chromatography tags can be used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag. Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Protein tags can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging). Tags can be combined, in order to connect proteins to multiple other components. However, with the addition of each tag comes the risk that the native function of the protein may be abolished or compromised by interactions with the tag. Therefore, after purification, tags are sometimes removed by specific proteolysis (e.g. by TEV protease, Thrombin, Factor Xa or Enteropeptidase).

Exemplary tags comprise the following, among others known to persons skilled in the art: Peptide tags, such as: AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE (SEQ ID NO:24)); Calmodulin-tag, a peptide that can be bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO:25)); polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE (SEQ ID NO:26)); E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR (SEQ ID NO:27)); FLAG-tag, a peptide recognized by an antibody (DYKDDDDK (SEQ ID NO:28)); HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA (SEQ ID NO: 29)); His-tag, typically 5-10 histidines that can be bound by a nickel or cobalt chelate (HEIHEIHH (SEQ ID NO:30)); Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL (SEQ ID NO:31)); NE-tag, a novel 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES (SEQ ID NO:32)) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS (SEQ ID NO:33)); SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:34)); Softag 1, for mammalian expression (SLAELLNAGLGGS (SEQ ID NO:35)); Softag 3, for prokaryotic expression (TQDPSRVG (SEQ ID NO:36)); Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK (SEQ ID NO:37)); TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC (SEQ ID NO:38)); V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST (SEQ ID NO:39)); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK (SEQ ID NO:40)); Xpress tag (DLYDDDDK (SEQ ID NO:41)); Covalent peptide tags such as: Isopeptag, a peptide which binds covalently to pilin-C protein (TDKDMTITFTNKKDAE (SEQ ID NO:42 SpyTag, a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK (SEQ ID NO:43)); SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK (SEQ ID NO:44)).

In embodiments described herein, any of the tags of SEQ ID NO:24-44, and other tags known to those skilled in the art, can comprise one or more amino acid substitutions, insertions, or deletions that do not alter the function of the tag, and can further comprise one or more additional amino acids, up to a maximum tag length of 100 amino acids. In preferred embodiments, the tag comprises up to a maximum of 20 amino acids in length.

In some embodiments, the protein tag can be a polyhistidine tag. A polyhistidine-tag is an amino acid motif in proteins that typically consists of six histidine (His) residues typically, often at the N- or C-terminus of the protein. It is also known as hexahistidine-tag, 6× His-tag, His6 tag and by the trademarked name His-tag (registered by EMD Biosciences). The total number of histidine residues can vary in the tag. N- or C-terminal his-tags can also be followed or preceded, respectively, by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endopeptidases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. Fluorescent hexahistidine CyDye tags are also available. These use Nickel covalent coordination to EDTA groups attached to fluorophores in order to create dyes that attach to the polyhistidine tag. This technique has been shown to be effective for following protein migration and trafficking. This technique can also be effective in order to measure distance via Fluorescent Resonance Energy Transfer.

In some embodiments, engineered microcompartment proteins comprise tags up to 8 amino acids in length inserted within the engineered microcompartment proteins as described herein. Exemplary tags include peptide tags such as AviTag, E-tag, FLAG-tag, His6-tag, Strep-tag and as well as other known to persons skilled in the art.

[00193]M some embodiments, the tags can be inserted within the A-domain of the encapsulin protein. In particular, the tags can be inserted within 1-8 residues adjacent to the N-terminus of the A-domain of SEQ ID NO: 47 (see Example 12 and 18). In some embodiments, the tags can be inserted in the E-loop.

In some embodiments herein described, an insertion region comprising at least one first protease cleavage site and the insertion region comprising the at least one second protease cleavage site have independently lengths up to 22 amino acids including any linker, protease cleavage sites and tags.

The term “linker” as used herein indicates a short peptide sequences that occur between protein domains. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. In particular, in engineered microcompartment protein of the disclosure linkers are typically peptide of 2 to 5 residues in combination with a protease cleavage site, a target protein, and/or a tag as will be understood by a skilled person upon reading of the present disclosure. The linker between the protease cleavage site and the encapsulin protein can comprise at least one glycine residue. The linker can be as short as two amino acids in length.

Exemplary linkers include GGGGS (SEQ ID NO:75), GSGSG (SEQ ID NO:76), GGGG (SEQ ID NO:77), GGG(SEQ ID NO:78), GG(SEQ ID NO:79), GS(SEQ ID NO:80), GSGS(SEQ ID NO:81), GGGS(SEQ ID NO:82), GGS(SEQ ID NO:83), GTS (SEQ ID NO:84), GGGGT (G₄T) (SEQ ID NO: 85) and others identifiable by a person skilled in the art.

In some embodiments wherein a target protein is packaged within a cage-forming encapsulin construct, the presence of a linker between the protease cleavage site and the N-terminus of the engineered encapsulin is preferred. In some embodiments insertion of a linker of at least 5 residues in length containing at least 1 glycine residue between the protease cleavage site and the encapsulin protein can facilitate the release of the target protein following the protease digestion (Example 19).

In some embodiments, the engineered microcompartment proteins can be designed to be cage forming engineered microcompartment proteins or non cage forming engineered microcompartment proteins as described herein.

Preferred cage forming engineered microcompartment proteins typically comprise one protease cleavage site between the C-terminus of the peptide and N-terminus of the engineered encapsulin. Preferred cage forming engineered microcompartment proteins typically also comprise a linker between the first protease cleavage site and the N-terminus of the engineered encapsulin wherein the linker is preferably flexible (containing at least 1 glycine residue) and greater than 2 amino acids in length. A typical linker is a penta-glycine linker (GGGGG SEQ ID NO: 143). The linker length is selected not to exceed a length that would cause the (peptide)+(protease cleavage site)+(linker) to be >80 amino acids. Preferred cage forming engineered microcompartment proteins further comprise one protease cleavage site within the loop region of the E-loop domain. This region is the unstructured region between beta-strands β2 and β3. Examples are insertions following residues V57 and D60 in T. maritima encapsulin. An additional protease cleavage site can also be placed in the A-domain within the beta-strand β5 and the subsequent unstructured region. An example is insertion following residue K138 in T. maritima encapsulin. The addition of this site may improve the kinetics of peptide release.

Preferred cage forming microcompartment proteins can be advantageously used for example when production of a protease-sensitive peptide where the cage protects the peptide from proteolysis during expression; storage of a peptide to protect it from proteolysis within cells or in vitro as purified protein are desired.

Preferred cage forming microcompartment proteins are also expected to be advantageously used when improved secondary structure formation (e.g., disulfide bond formation) during peptide expression is desired.

Preferred non-cage forming engineered microcompartment proteins typically comprise one protease cleavage site between the C-terminus of the peptide and N-terminus of the engineered encapsulin. Preferred non-cage forming engineered microcompartment proteins typically also comprise a linker between the first protease cleavage site and the N-terminus of the engineered encapsulin. The linker is preferably flexible (containing at least 1 glycine residue) and can be as short as 2 amino acids in length. The linker is selected not to exceed a length that would cause the (peptide)+(protease cleavage site)+(linker) to be >80 amino acids. Preferred non-cage forming engineered microcompartment proteins typically further comprise one protease cleavage site in beta-strand β3 of the E-loop domain, within 9 amino acids of the N-terminus of the adjacent P-domain fragment. An example is an insertion following residue K71 in T. maritima encapsulin. An additional protease cleavage site followed by an affinity purification tag can also be placed in the A-domain within the beta-strand β5 and the subsequent unstructured region. An example is insertion following residue K138 in T. maritima encapsulin. Here, the affinity tag can be used for purification of the construct, which is accessible in a non-cage forming encapsulin mutant.

Preferred non-cage forming microcompartment proteins can be advantageously used for example when_high yield production and release of a peptide—purification shall be done rapidly and in the presence of protease inhibitors to prevent any proteolysis. Non-cage forming microcompartment proteins are also expected to be advantageous when_an improved secondary structure formation (e.g., disulfide bond formation) during peptide synthesis is desired.

A skilled person will be able to identify how to configure an engineered microcompartment protein of the disclosure based on the target protein and other features of the related production. For example, in some embodiments wherein the target protein is protease-sensitive, the insertion of the one or more protease sites within the encapsulin shell protein is preferably selected to provide a cage forming engineered microcompartment protein to maximize incorporation of expressed peptide into an encapsulin cage such that the cage-forming encapsulin construct can protect the target protein from proteolysis during expression or for the storage of the expressed target protein within cells or in vitro as purified protein. The cage-forming encapsulin construct is also expected to facilitate improved secondary structure formation, such as disulfide bond formation, during peptide expression.

In some of these embodiments, the protease cleavage site can be preferably provided within the unstructured loop region of the E-loop domain. In some exemplary embodiments exemplified in Example 18 the protease cleavage site can be inserted following residues V57 and D60 in T. maritima encapsulin. Insertion of a protease site following K138 in T. maritima (corresponding to the region within 2-24 amino acids of the N-terminus of the A-domain (the A-domain within the beta-strand β5 and the subsequent unstructured region) also maintains cage formation.

In embodiments, wherein the target protein is protease-sensitive, protease cleavage sites can be selected to provide a non-cage forming engineered microcompartment protein in embodiments wherein purification of the non-cage forming engineered microcompartment protein is performed under conditions to prevent proteolysis, such as in the presence of additives that prevent proteolysis such as B-PER II detergent or protease inhibitors. In addition, or in the alternative purification can be performed within the timeframe when degradation of less 50% of the protein is detected, typically within 16 hrs. or less. Methods to detect protein degradation comprise densitometry performed on Western blot or SDS-PAGE of the proteins in the lysate comprising the protein and additional techniques identifiable by a skilled person. In some of those embodiments, a protease cleavage site can be placed at the C terminus of E-Loop domain or within 1-8 amino acids adjacent to the C-terminus of the E-loop domain within beta-strand β3. An exemplary embodiment is an insertion following residue K71 in T. maritima encapsulin (see Example 18).

In some embodiments wherein fast release of the target protein is desired, the insertion of the one or more protease sites within the encapsulin shell protein is preferably selected to provide a non-cage forming engineered microcompartment protein comprising at least one protease cleavage site in the beta-strand β3 of the E-loop domain or in combination with at least one protease cleavage site in the unstructured region of the E-Loop and/or the A domain within the beta-strand β5 and the subsequent unstructured region. The second insertion can improve the kinetics of peptide release in some embodiments. An exemplary embodiment is provided by constructs comprising a protease cleavage site following residue K71 in T. maritima encapsulin (see Example 18).

In some of these embodiments wherein a high yield production and release of a target peptide is desired, a protease cleavage site can be selected within 0-8 amino acids residues adjacent to the C-terminus of the E-loop of the encapsulin protein within the beta-strand β3. In an exemplary embodiment the protease cleavage site is inserted following residue K71 in T. maritima encapsulin (Example 18). Such insertion site can effectively disrupt cage formation (see Example 19).

In several embodiments, the non-native protein to be produced or provided with methods of the disclosure comprise proteins or peptides that can be used as chemotherapeutic drugs in treating cancer to kill, inhibit growth or halt the replication and/or spread of cancerous cells in a patient. In some of those embodiments, the non-native protein or peptides are AMPS that can be used in cancer treatment.

In methods and systems herein described and related cell and compositions, one or more target proteins and in particular one or more proteins non-native to a bacterial cell are expressed in said cell within at least one engineered microcompartment protein to form at least one engineered microcompartment comprising the one or more toxic non-native proteins within the microcompartment.

The term “express” as used herein with reference to proteins or peptide indicates the way in which proteins or peptides are synthesized, modified and regulated in living organisms. Typically, protein expression includes DNA transcription, RNA processing, translation, and post-translational modification of a protein as will be understood by a skilled person. In particular, the term protein expression refers the process of generating a specific protein within a cell and includes the transcription of the recombinant DNA to messenger RNA (mRNA) and the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations

Expression system for protein production comprise a combination of an expression vector, its cloned DNA, and the host cell for the vector that provide a context to allow a non-native gene function in a host cell, that is, produce proteins. Example expression systems are 1) BL21(DE3) host cells that express protein from an expression vector that contains a pT7 phage promoter; and 2) BL21 host cells that express protein from expression vectors that contain pT5 or pRha promoters. Additional expression systems and related host cells, vector and promoters are identifiable by a skilled person

The term “cell” or “ bacterial cell” as used herein indicates a bacterial cell with bacteria indicating several prokaryotic microbial species which include but are not limited to Gram-positive bacteria, Proteobacteria, Cyanobacteria, Spirochetes and related species, Planctomyces, Bacteroides, Flavobacteria, Chlamydia, Green sulfur bacteria, Green non-sulfur bacteria including anaerobic phototrophs, Radioresistant micrococci and related species, Thermotoga and Thermosipho thermophiles. More specifically, the wording “Gram positive bacteria” refers to cocci, nonsporulating rods and sporulating rods, such as, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus and Streptomyces. The term “Proteobacteria” refers to purple photosynthetic and non-photosynthetic gram-negative bacteria, including cocci, nonenteric rods and enteric rods, such as, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema and Fusobacterium. Cyanobacteria, e.g., oxygenic phototrophs.

In embodiments herein described cytotoxic proteins are expressed inside engineered microcompartment proteins, the engineered microcompartment proteins can shield the cells from toxicity of the cytotoxic proteins, allowing the cells to grow and thus produce more cytotoxic proteins.

In some embodiments, methods of the present disclosure comprises introducing into the cell at least one polynucleotide encoding at least one engineered microcompartment protein herein described, the at least one polynucleotide operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different microcompartment proteins to form at least one empty microcompartment within the cell.

As used throughout, “regulatory elements” are regions of non-coding DNA which regulate the transcription of nearby genes. Examples of regulatory elements are promoters and enhancers. Enhancers are regions of DNA that can be bound with proteins (activators) to activate transcription of a gene or transcription. Promoters are regions of DNA that initiate transcription of a particular gene. In the embodiments described, types of promoters used are over-expression promoters, low-level promoters and tunable promoters. Tunable promoters are not constitutive and can be activated or inactivated as a result of culturing conditions and/or additional elements. In some embodiments, tunable promoters are activated in the presence of a compound introduced into the culture media. Examples of tunable promoters includepRha. In the embodiments described, selection of a promoter is determined by several factors including, but not limited to, the nature of the protein being expressed and the desired expression level of the expressed protein. In the embodiments described, low-level promoters are used when the toxic non-native protein is not efficiently localized to the interior of a microcompartment so as to reduce toxicity to a cell from accumulation of the toxic non-native protein in the cell. In the embodiments described, the use of tunable promoters is used to express a protein at a certain level and/or time during culturing of the cell. In the embodiments described, the type of promoter used is influenced by the interplay between the microcompartment proteins and the toxic non-native proteins.

Accordingly, selection of the appropriate regulatory elements and the at least one polynucleotide can be performed with procedures identifiable by a skilled person.

As used throughout, “operably linked” is defined as a functional linkage between two or more elements. In particular, the term “operably linked” or “operably connected” indicates an operating interconnection between two elements finalized to the expression and translation of a sequence. Functional linkages between elements in the sense of the present disclosure are identifiable by a skilled person. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) comprises a functional link that allows for expression of the polynucleotide of interest. Another example of operable linkage is provided by a control sequence ligated to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked elements are contiguous or non-contiguous and comprise polynucleotides in a same or different reading frame. Additionally, “operably linked” refers to proteins that are linked together wherein the linkage does not impact the function of the individual proteins.

In embodiments herein described, the engineered microcompartment protein comprises encapsulin or encapsulin-like proteins fused with a toxic non-native protein at the N-terminus of the encapsulin and inserted with one or more protease cleavage sites described herein. In some embodiments, one or more protein tags can be added through genetic modification of the engineered microcompartment proteins herein described in accordance with the present disclosure.

In some embodiments an engineered microcompartment protein can be introduced in a cell to form an engineered microcompartment according to a method to provide an engineered microcompartment protein in a bacterial cell herein described. The method comprises introducing into the cell at least one polynucleotide encoding at least one engineered microcompartment protein herein described. The at least one polynucleotide operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the cell, the at least one microcompartment protein capable of assembling with one or more same and/or different microcompartment proteins to form at least one microcompartment within the cell. The assembled engineered microcompartment proteins encompass an interior target protein.

As used throughout, “introducing into the cell” with respect to the polynucleotides refers to inserting a polynucleotide encoding a protein or peptide into a cell or population of cells. One of ordinary skill in the art can readily appreciate that a variety of methods can be used to achieve this such as transformation, transfection, viral transduction and/or injection. In the embodiments described, successful introduction of a polynucleotide into a cell can be assessed by selecting for cells that have taken up the polynucleotide. This is done, for example, by incorporating into the polynucleotide an antibiotic resistance marker against an antibiotic that a cell is typically sensitive to. In some embodiments described, ampicillin resistance genes and kanamycin resistance genes are used on polynucleotides to assess positive insertion of a polynucleotide into a bacterial cell sensitive to ampicillin and kanamycin. Following insertion of a polynucleotide carrying an ampicillin resistance gene, for instance, cells are grown in media containing ampicillin to select for cells that have successfully taken up the polynucleotide.

Introduction of a polynucleotide can be performed for example by chemical transformation or electroporation or other methods identifiable by a skilled person. In some exemplary embodiments performed in E. coli, chemical transformation can be performed by incubating CaCl₂-treated E. coli cells with the plasmid(s) of interest and heat shocking the cells at 42° C. for an appropriate time period (<60 s) to encourage the cells to take up the plasmid(s). Cells are then diluted with rich medium and incubated at 37° C. to allow for heat shock recovery and expression of one or more antibiotic resistance genes. Cells are then plated on solid-agar medium supplemented with the appropriate antibiotic to select for cells that have taken up the polynucleotide. Similarly, in electroporation, E. coli cells are incubated with the polynucleotide of interest and electroporated at an appropriate voltage to increase cells uptake of the polynucleotide as will be understood by a skilled person. Subsequent steps are the same as for chemical transformation.

In some embodiments, introducing into the cell the at least one polynucleotide can be performed by introducing an expression vector comprising the at least one polynucleotide and the one or more first regulatory elements in a configuration leading to transcription of the engineered microcompartment protein carried on the expression vector.

As used throughout, an “expression vector” is a plasmid or virus designed for protein expression in cells. The expression vector is used to introduce a specific gene into a target cell and uses the cell's mechanism for protein synthesis to produce the protein encoded by the gene. In the embodiments described, genes delivered include toxic non-native proteins (such as Ap and HBCM-2) and microcompartment proteins (such as encapsulin of SEQ ID NO: 47). In the embodiments described, a plasmid is engineered to contain regulatory sequences that act as enhancer and/or promoter regions and lead to efficient transcription of the gene carried on the expression vector. Such plasmids also contain selection markers, such as antibiotic resistance markers, to select for cells that have successfully taken up the plasmid. The plasmids herein used for the expression of the engineered microcompartment can also comprise linker sequences, tags and protease cleavage sites. Examples of constructs are pMCY124, pMCY125, and pMCY133 (see Example 12-15).

In order to introduce the engineered microcompartment proteins to a cell, one or more genes encoding the appropriate engineered microcompartment proteins can be cloned and placed under the control of a promoter (constitutive or inducible) in a given plasmid or other vector of interest containing an antibiotic resistance marker. Genes for coding the non-native protein and encapsulin protein can be place in tandem behind a given promoter with appropriate ribosomal re-initiation sites to ensure all proteins are expressed. The plasmid containing one or more engineered microcompartment genes is then transformed into the host organism by either chemical transformation or electroporation. In the case of a constitutive promoter, microcompartment protein is expressed from the plasmid constantly during growth of the host organism. In the case of an inducible promoter, microcompartment protein is expressed from the plasmid by addition of inducer to growth medium (e.g. IPTG, rhamnose).

In some embodiments, the one or more first regulatory elements comprise an over-expression promoter, a low-level constitutive promoter or a tunable promoter. For example, in some embodiments, the one or more first regulatory elements can comprise a T7, pTet, pRha or pT5 promoter.

In some embodiments, the engineered microcompartment proteins can be designed and altered to support functional expression of the cytotoxic non-native proteins to be encapsulated inside one or more engineered microcompartment. For instance, the expression system can be engineered to over-express the encapsulin shell proteins and the non-native proteins to improve yields, thus allowing for higher expression of encapsulated non-native proteins.

In some embodiments, where the engineered microcompartment proteins are to be overexpressed, expression of one or more appropriate engineered microcompartment proteins can be placed under the control of a highly inducible promoter (e.g., T7, T5, rhamnose). The engineered microcompartment proteins can also be over-expressed from a high copy number plasmid containing an appropriate origin (e.g., pUC) in order to ensure multiple copies of the appropriate genes are expressed. The nucleotide sequence of the microcompartment protein genes can be optimized based on host organism codon usage in order to achieve overexpression as well as according to approaches such as the ones described in reference ([24]) and other approaches identifiable by a skilled person.

In some embodiments herein described, one or more genes for toxic non-native proteins are fused to one or more genes for encapsulin proteins under the control of an inducible promoter in a given plasmid or other vector of interest to form the at least one polynucleotide encoding an engineered microcompartment protein. The plasmid (or other vector) containing the polynucleotide for the engineered microcompartment protein can be transformed into a host cell by chemical transformation or electroporation and selected for using antibiotic resistance markers or other markers identifiable by a skilled person.

In some embodiments, at least one polynucleotide comprises one or more polynucleotides encoding for two or more microcompartment proteins.

In some embodiments, the at least one polynucleotide comprises one or more polynucleotides encoding for encapsulins (see Examples 12-16).

In some embodiments, the at least one polynucleotide comprises one or more polynucleotides encoding for one or more of non-native proteins. The non-native protein insertions comprise up to 80 amino acids in length (see Examples 15-17).

In some embodiment herein described, the genes encoding for toxic non-native proteins are operably linked to one another and/or to the genes encoding for encapsulin proteins through a linker. The linker between the non-native protein genes and between the non-native protein gene and the encapsulin gene is configured not to impact the expression of the polynucleotide and the proper folding of the formed engineered microcompartment proteins. The linker also comprises a protease cleavage site specific to a protease, thus allowing the release of the toxic non-native proteins from the encapsulin proteins by proteolysis.

In some embodiments, the at least one polynucleotide encoding the engineered microcompartment also comprises one or more protease cleavage sites inserted within the genes encoding encapsulins to ensure full digestion of the encapsulin cage and thus full release of the non-native proteins (Examples 12-17).

In some embodiments, two or more polynucleotides can be introduced in combination, simultaneously or sequentially. Whether to introduce two or more polynucleotides in combination, sequentially or simultaneously depends on the nature of the proteins being expressed from the polynucleotides and the desired results. In the embodiments described, polynucleotides are expressed sequentially, for instance, so as to effectively select for positive insertion of the polynucleotides. For instance, a first polynucleotide encoding for a microcompartment protein and containing an ampicillin resistance gene can be introduced into a group of cells that are sensitive to ampicillin and kanamycin. Following insertion of the first polynucleotide, the cells are cultured in the presence of ampicillin to select for those that have taken up the first polynucleotide. Next, these cells are introduced to a second polynucleotide encoding for a toxic non-native protein and containing a kanamycin resistance gene. Following insertion of the first polynucleotide, the cells are cultured in the presence of kanamycin to select for those that have taken up the second polynucleotide. The resulting cells are thus selected for successful incorporation of both polynucleotides. A similar strategy is taken when the second polynucleotide encodes for a protein that is extremely toxic to the cells and/or inefficiently localized to the interior of a microcompartment. In such an example, a first polynucleotide encoding for a microcompartment protein is introduced before the second polynucleotide encoding for the toxic protein so as to prevent the cell from toxicity following expression of the protein from the second polynucleotide.

In the embodiments described herein, polynucleotides introduced into a cell encode for a single protein or peptide or several proteins or peptides that function together.

As used throughout, “conditions” for culturing the cells refer to the various elements required to select and/or maintain cells as well as to the various elements required to obtain the desired amount of protein expression from the polynucleotides. Elements required for these purposes include culture media, antibiotics, chemical inducers to promote expression from a promoter (e.g., i sopropyl-B-D-thiogalactopyranoside, rhamnose, arabinose), CO₂ concentrations, temperature, agitation (in rotations per minute, rpm) and additional factors required to ensure that the proteins are expressed; other elements would be readily appreciated by one of ordinary skill in the art. Additionally, elements include factors that are required for the expressed proteins to function as intended.

In embodiments herein described, the methods further comprises introducing into the cell at least one second polynucleotide encoding one or more proteases, each protease capable of cleaving at a protease cleavage site inserted within the engineered microcompartment protein, thus releasing the non-native protein from the engineered microcompartment protein. The at least one second polynucleotide is operably linked to one or more second regulatory elements leading to the expression of the at least one protease.

Exemplary proteases include Human Rhinovirus (HRV) 3C Protease, Enterokinase, Factor Xa, Tobacco etch virus protease (TEV protease), Thrombin and others known to a person skilled in the art.

In some embodiments, introducing into the cell the at least one second polynucleotide encoding for the proteases is performed by introducing an expression vector comprising the at least one polynucleotide of the at least one second polynucleotide and the one or more second regulatory elements in a configuration leading to transcription of the protease carried on the expression vector.

In some embodiments, the one or more second regulatory elements comprise a promoter, a low-level constitutive promoter or a tunable promoter. In some embodiments, the one or more second regulatory elements comprise an enhancer.

In some of those embodiments, the one or more second regulatory elements are different from the one or more first regulatory elements operably linked to the polynucleotide encoding the engineered microcompartment protein. In some embodiment, the second regulatory elements in the polynucleotide encoding the proteases comprise a pRha or pT7 promoter while the first regulatory elements in the polynucleotide encoding the engineered microcompartment proteins comprise pTet or pT5.

In particular, in embodiments herein described, introducing the second polynucleotide is performed in combination with the introducing of the first polynucleotide to obtain the toxic non-native protein within the cell.

In some embodiments, proteases can be added directly to the lysed cells or purified engineered microcompartment proteins to release the non-native protein from the microcompartment proteins. In some of these embodiments, the method further comprises purifying the at least one engineered microcompartment protein and adding to the purified engineered microcompartment protein at least one protease targeting the protease cleavage sites of the engineered microcompartment protein to release the non-native target protein from the engineered microcompartment protein to obtain the non-native target protein.

The target protein can be purified by size exclusion chromatography or by a centrifugal filter with an appropriate molecular weight cutoff in order to separate the target protein from the microcompartment protein based on size. Alternatively, ion exchange chromatography or reverse phase (e.g., C18) chromatography can be used when appropriate as will be understood by a skilled person.

Purification of the at least one engineered microcompartment protein can be performed rapidly (within 16 hrs. e.g. within 4 h or less) and/or in the presence of suitable additives and/or protease inhibitors to prevent any proteolysis of the engineered microcompartment proteins at this stage. In some other embodiments, the at least one protease can be added to lysed cells expressing the engineered microcompartment proteins herein described. In some embodiments, lysis of the cells can be performed rapidly within 4 h or other suitable time in view of the reaction mixture. Rapid purification of the microcompartment or rapid lysis of the cells are particularly preferred for non cage forming engineered microcompartment protein comprising a protease sensitive target protein as will be understood by a skilled person.

The term “protease-sensitive” as described herein indicates proteins that are targeted for proteolytic degradation by native proteases in the host cell or cell free reaction where the expression of the protein is performed.

In embodiments in which an engineered microcompartment protein comprises a proline between the N-terminus of the first at least one protease cleavage site and the C terminus of the target protein the method can further comprise, contacting the purified non native target protein with a carboxypeptidase to allow reaction of the carboxypeptidase with the carboxy terminal residues of the purified non-native target protein.

First, different protease cleavage sites between the C-terminus of the peptide and the N-terminus of the engineered encapsulin should be tested to determine if there is a residual protease cleavage site that does not interfere with peptide activity.

These additional contacting of the purified target protein can be performed to achieve activation of a non-active peptide to an active peptide or improvement of the activity of an active peptide.

In some embodiments, the present disclosure provides a method to express significant amounts of non-native, cytotoxic proteins in a host organism for isolation and production purposes. The method can be applied to proteins of interest that are difficult to produce due to their cytotoxicity to the host organism. In an exemplary method, cytotoxic, non-native proteins are engineered to be encapsulated in encapsulin cage in E. coli in order to prevent their cytotoxicity. As proof of this concept, in some examples herein described, the cytotoxic non-native proteins Ap from honey bee were fused to encapsulins to form engineered microcompartment proteins in the cytoplasm of E. coli, thus shielding the toxicity of Aps from the cells as described in Example 12-15 and 24. In some other examples, the cytotoxic non-native HBCM-2 peptides were fused to encapsulins to form engineered microcompartment proteins thus shielding the toxicity of the peptides from the cells (Example 16-17).

In some embodiments, the engineered microcompartment proteins formed by fusing cytotoxic target proteins, such as non-native HBCM-2 and AP, to encapsulins can achieve a robust expression while remaining less susceptible to proteolysis in comparison with fusion proteins of the same cytotoxic non-native peptides with other common carrier proteins (Example 24). In particular, the engineered microcompartment proteins formed by fusing cytotoxic non-native target proteins to encapsulins also confer limited toxicity to the host cells even at a high over-expression level (Example 23).

In some embodiments, the methods herein described can produce a large amount of active, cytotoxic non-native proteins in vivo or in vitro (following cleavage from the engineered microcompartment protein) having the same level of activity as the chemically synthesized proteins (see Example 22). In some embodiments, the present disclosure addresses issues associated with production of non-native, cytotoxic proteins in the host organism E. coli, and can be associated with in vitro purification systems (cell free expression, as described in the www webpage lifetechnologies.com) excretion tags excreting the cytotoxic proteins from E. coli (as described in the www webpage dna20.com), which are used to produce cytotoxic proteins.

In some embodiments, methods and systems of the disclosure herein described can be used express cytotoxic proteins directly in bacterial microcompartments to minimize toxicity, and/or reduce problems associated with proper folding and secretion associated with secretion tags. The methods, systems and related compositions and cells of the disclosure can result in several embodiments with reduced costs and higher production levels compared to those of prior methods.

In some embodiments, engineered microcompartment proteins, related engineered microcompartment and related vectors, cells, compositions, methods and systems can be used to introduce in a same engineered microcompartment a same or different target protein.

An exemplary illustration of steps of methods to provide one or more target proteins such as toxic or non-toxic non-native proteins herein described and in particular Ap are illustrated with reference to exemplary engineered microcompartment proteins expressed in E. coli cells through use of specific regulatory sequences as will be understood by a skilled person.

A first fusion gene encoding an engineered microcompartment protein is created, the fusion gene comprising a gene of the desired non-native protein fused to the N-terminus of a gene encoding an encapsulin protein. Protease cleavage site such as TEV or thrombin can also be placed between the non-native protein and the encapsulin protein to later cleave off the non-native protein. Protease cleavage sites are also placed within the encapsulin proteins to enable digestion of the encapsulin and thus release of the non-native protein.

The fusion gene is then placed under the control of an inducible promoter in a plasmid of interest, which is then transformed into an E. coli expression strain (e.g., C43 cells). The transformed E. coli cells then grow in an appropriate medium (LB medium) at an appropriate temperature.

A second gene encoding a protease can be created and co-expressed with the fusion gene encoding an engineered microcompartment protein.

The cells are induced for engineered microcompartment protein formation using the inducer for the plasmid containing the microcompartment proteins (e.g., IPTG for the pT5 promoter).

Alternatively, instead of co-expression of the fusion gene and the protease-encoding gene, proteases can be added directly to lysed cells expressing the engineered microcompartment protein or to purified engineered microcompartment protein.

In order to isolate the toxic protein of interest inside microcompartments, the cells are harvested after desired induction time and resuspended in an appropriate buffer. The cells are then lysed by an appropriate method including sonication, French press lysis, detergent lysis with lysozyme and other methods identifiable to a person skilled in the art. The cell debris can be removed by centrifugation at 12,000 g, 4° C. for 10 min and supernatant can be collected.

To collect the toxic protein of interest, appropriate detergent is added to the soluble fraction if necessary (e.g., Empigen BB) and incubated for sufficient time to solubilize desired protein. The soluble fraction is then loaded on an affinity chromatography column. The column is washed and the desired protein is eluted with the appropriate buffers. The fractions containing the desired protein can then be collected and stored as necessary.

Engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be applied broadly to other cytotoxic proteins for expression and purification from bacterial cells by replacing the gene for Ap with the gene for a cytotoxic protein (toxic, non-native protein) of interest and by replacing the medium and regulatory sequences of E. coli with the ones of a desired bacteria as will be understood by a skilled person. Thus, the methods, systems, cell and compositions of the disclosure have wide applications in the biosciences, where this novel technology could be used for the efficient production of proteins that are normally difficult to produce.

In embodiments herein described, engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be used to produce a toxic non-native protein, by expressing the toxic non-native protein within BMC in a bacterial cell and by isolating the toxic non-native protein from the bacterial cell.

In some embodiments, engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be used to shield the toxicity of pathway intermediates and increase reaction efficiencies in nature. In those embodiments, a toxic non-native protein can be expressed within a BMC and additional molecule forming the pathway can be provided within the cell. In those embodiments, small molecule substrates and products of the enzymes can passively diffuse in and out of the BMCs via pores in the shell proteins, while pathway intermediates remain sequestered inside the BMCs. In such case, not only the toxic pathway intermediates can be shielded from the host organism, but the local concentration of enzymes and substrates also increases, leading to improved reaction efficiency.

In several embodiments, the present disclosure provides engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure that provide a bioengineering application of microcompartments that has not been previously explored.

In some embodiments, a bacterial cell obtainable by any one of the methods of the disclosure is described, and in particular a cell comprising at least one toxic non-native protein within at least one engineered microcompartment within the cell.

In some embodiments, a bacterial cell herein described further comprises various toxic non-native proteins wherein the various non-toxic proteins function and/or aggregate independently of or in combination with one another.

In some embodiments, in a bacterial cell herein described the various non-toxic proteins reside within the at least one microcompartment within the cell either independently of or in combination with one another.

In some embodiments, in a bacterial cell herein described various non-toxic proteins reside within at least two or more microcompartments within a cell.

In some embodiments, in bacterial a cell herein described at least one of the microcompartments comprises at least one additional component.

In some embodiments, in a cell herein described the at least one additional component is presented to the at least one toxic-nonnative protein.

In some embodiments, a composition is described comprising one or more bacterial cells obtained from any one of the methods of the disclosure, and/or by any one of the systems of the disclosure and/or any one of the cells herein described together with a suitable vehicle.

In some embodiments, the engineered microcompartment proteins, and related target proteins, insertion regions, tags. Linkers, engineered microcompartments, bacterial cells herein described can be comprised in a composition together with a suitable vehicle. The term “vehicle” as used herein indicates any of various media acting usually as solvents, carriers, binders or diluents for the non-native toxic proteins and/or related cells that are comprised in the composition as an active ingredient. In particular, the composition including the non-native toxic proteins and/or related cells can be used in one of the methods or systems herein described.

As disclosed herein, the engineered microcompartment proteins, the target proteins, insertion regions, tags, linkers regulatory sequences, vectors and/or related cells herein described can be provided as a part of systems to produce one or more non-native toxic proteins, and in particular can be used in methods to produce or provide a non-native toxic protein herein described. The systems can be provided in the form of kits of parts. In a kit of parts, the non-native toxic proteins, regulatory sequences, vectors and/or related cells and other reagents to produce or provide a non-native toxic protein can be comprised in the kit independently. The non-native toxic proteins, regulatory sequences, vectors and/or related cells can be included in one or more compositions, and each component can be in a composition together with a suitable vehicle.

Exemplary components of a kit of parts and of constructs herein described comprise the nucleotide sequence of the linkers, protease cleavage sites, and histidine affinity tags that are codon optimized for maximum expression in E. coli such as the one described in the Examples section. Additional components can include labeled molecules and in particular, labeled polynucleotides, labeled antibodies, labels, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure. The terms “label” and “labeled molecule” as used herein as a component of a complex or molecule referring to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like.

In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the assay, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).

Further details concerning methods and system, cells and compositions of the present disclosure will become more apparent hereinafter from the following detailed disclosure of examples by way of illustration only with reference to an experimental section.

EXAMPLES

The engineered microcompartment proteins herein described and related engineered microcompartments, methods and systems for engineering bacterial cells, as well as bacterial cells herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary methods and systems for expressing non-native, cytotoxic proteins or pathways involving these proteins in engineered microcompartment proteins for synthetic biology applications. The following examples demonstrate that engineered microcompartment proteins are useful platforms to produce in a bacterial cell proteins that are toxic and/or degradable in the cell minimizing cytotoxicity and degradation while improve efficiency of non-native protein expression compared to other convention approach for protein expression. The development of this platform can be broadly used in various fields including biofuels, biopharmaceuticals, biodefense, bioremediation as well as many other applications in bioscience in general.

A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional non-native proteins and bacterial systems and related methods and systems according to embodiments of the present disclosure.

Example 1 Exemplary AMPs

Exemplary AMP used in some experiments herein described are listed in Table 1.

TABLE 1 Amino acid sequences of exemplary AMP AMP peptide Amino acid sequence SEQ ID NO HBCM2 KWKSFIKKLTKAAKKVVTTAKKPLIV 9 HBCM3 KWKKFIKSLTKSAAKTVVKTAKKPLIV 10 Cecropin PR- RRRPRPPYLPRPRPPPFFPPRLPPRIP 11 39 PGFPPRFPPRFP Apidaecin Ia GNNRPVYIPQPRPPHPRI 12

HBCM2 and HBCM3 are two AMPs that have activity against antibiotic-resistant P. aeruginosa. These two AMPs are optimized hybrid peptides of moth cecropin and bee melittin [18, 19]. These peptides also have been shown to have anti-inflammatory activity in cystic fibrosis mouse models [19]. Non-lytic AMPs cecropin PR-39 and apidaecin Ia, which are derived from pig and honeybee, respectively [25, 26] are also expressed. Collins and coworkers have shown that these two AMPs have high bactericidal activity but do not induce lysis of pathogens, avoiding release of endotoxins that may be toxic to the human host [27]. Thus, they may be better suited for therapeutic applications. All four of these AMPs (Table 1) lack cysteine residues and thus do not require disulfide bond formation for activity, making them more amenable to heterologous expression [28]. Other AMPs with activity toward P. aeruginosa based on the Antimicrobial Peptide Database can also be screened for expression [22].

Example 2 Encapsulation Strategies Using Encapsulins

Protein expression systems can be designed to express the AMPs encapsulated inside an encapsulin microcompartment system. In encapsulin systems (e.g., ferritin cages), a single, repeating encapsulin protein is responsible for encapsulating an interior protein, in a typical ratio of 60 encapsulins to 6 interior proteins [2, 29]. In contrast to other BMC systems, in which several different BMC shell proteins are required to encapsulate interior protein [30], encapsulin systems are less complex with a single encapsulating protein. They are therefore expected to have improved formation of compartments in a recombinant system, and thus higher loading of interior protein, compared to traditional BMC systems. These expression systems are initially designed under traditional inducible promoters (e.g., pTet, pT5) in order to test and optimize maximum production levels.

In order to target the AMPs to the interior of encapsulins, two potential strategies (FIGS. 9A-B) are employed. In the first strategy (FIG. 9A), the encapsulin shell protein will be directly fused to the C-terminus of the given AMP in one single polypeptide chain. The first strategy design is based on initial studies that suggest that interior protein-encapsulin fusions allow all interior proteins to be encapsulated by the encapsulin shells, without sacrificing the integrity of the formed encapsulin compartments. This fusion system is, furthermore, predicted to have more AMP molecules loaded into the encapsulin compartments (60:60 encapsulin:interior protein), compared to the native system (60:6 encapsulin:interior protein) [2], which should encourage higher production of AMPs in the recombinant system. The linker region between the interior protein and encapsulin can be optimized for efficient loading of AMPs into the encapsulins. Here, computational modeling can be performed to guide linker optimization for efficient loading into the compartments.

In some scenarios, the mechanism of action for AMP toxicity is carried out in the E. coli host before encapsulation occurs. This is a possibility since 60 units of the fusion protein will need to be synthesized to assemble one full encapsulin compartment, but only 1 unit of the fusion protein can cause toxicity. Thus, as an alternative to the first strategy, in the second strategy (FIG. 9B) a known C-terminal encapsulin targeting tag (18 amino acids, Encaptag) [2] will be fused to the AMP peptide and the AMP-Encaptag will be co-expressed with the encapsulin shell protein from two different promoter systems, pTet and pT5. In this system, the encapsulin shell proteins will be expressed first so that when the AMP-Encaptag protein is subsequently expressed, there will be sufficient encapsulin shell protein available for immediate AMP encapsulation. Toxicity shielding of AMPs and production amounts in either strategy will be determined using growth assays and SDS-PAGE/Western blots for quantification. If toxicity is shielded, better growth and higher production of AMPs can be expected in cells expressing AMP-encapsulin compared to AMP alone. Expression optimization will be performed to determine the maximum amount of encapsulated AMP that can be produced with limited toxicity to the host E. coli cells.

Example 3 Identification of Proteolysis Conditions

Once an expression system for encapsulating AMPs is established, conditions to release the AMPs from the interior of the encapsulins by proteolysis can then be developed. Here, proteolytical cleavage of AMPs from the encapsulin shell/Encaptag as well as cleavage of specific sites in the encapsulin shell protein can be applied so that the entire structure can be degraded for release of AMPS.

FIG. 11 illustrates the design of a protease-sensitive encapsulin. Antimicrobial peptide (AMP) is fused to the N-terminus of the encapsulin protein. Specific protease recognition sites are inserted between the AMP and encapsulin protein as well as within the encapsulin protein itself. The fusion protein is expressed from a T7 inducible promoter and purified. The purified fusion is digested with a specific protease that targets the protease recognition sites and the AMPS are released and thus can be isolated for a desired function.

Known protease cleavage sites (i.e., thrombin, enterokinase) [31] will be inserted between the AMP protein and encapsulin shell/Encaptag as well as within the encapsulin shell protein itself. Computation modeling will be used to identify potential cleavage insertion sites that would cause the encapsulin compartments to fall apart upon protease cleavage but would otherwise not affect the integrity of and loading into the compartments. Expression of AMP-encapsulin systems with appropriate protease cleavage sites will be tested for toxicity shielding and production amounts using growth assays and SDS-PAGE/Western blots. Microcompartments from stable AMP-encapsulin systems will be subsequently isolated using established differential centrifugation methods [2, 32] and treated with an appropriate protease to determine if AMP can be efficiently released from the compartments. Efficient release will be assessed by separation of AMP from encapsulin via size-exclusion chromatography or Ni-NTA affinity chromatography, where the AMP will be tagged with a His₆ tag that will not be cleaved from the AMP during proteolysis. If release of AMP is efficient, the AMP and encapsulin can be expected to purify as separate fractions during chromatography, whereas if release is inefficient, co-purification of the components is expected.

Recognition sequences and cleavage sites of exemplary proteases are shown in

TABLE 2 / forward slash (/) indicates where protease cleaves the protein sequence. Table 2: Recognition sequences and cleavage sites of exemplary proteases Sequence Enzyme Name and Cleavage SEQ ID NO Human Rhinovirus (HRV) LEVLFQ/GP 13 3C Protease Enterokinase DDDDK/ 14 Factor Xa IEGR/ 15 Tobacco etch virus ENLYFQ/G 16 protease (TEV protease) Thrombin LVPR/GS 17

Example 4 Release of AMPs from E. coli

Once an appropriate protease-sensitive AMP-encapsulin system is identified, experiments can be performed to test whether AMP can be released from encapsulin within the E. coli host organism and subsequently from the cell itself by co-expression of the AMP-encapsulin system and the appropriate protease.

In these studies, the AMP-encapsulin system will be expressed first from a pTet (and pT5 if necessary) promoter. Once high levels of AMP-encapsulin are produced, protease expression will be induced from a different promoter (i.e., pT7 or pRha). A lysis protein, such as colicin E7 lysis protein [33] may also need to be co-expressed with the protease to achieve efficient lysis of the bacterial cell to release the AMPs. The kinetics of AMP release from cells into the spent medium over time upon proteolysis/lysis induction will be assessed via SDS-PAGE/Western blot.

In order to compare the lysis system to known systems [33, 34], control strains of E. coli that 1) secrete the AMP candidates via YebF secretion signals [35] and 2) produce AMPs without encapsulin, will be generated, which are released by lysis. The rate and total amounts of AMP released will be compared among the control strains and the protease-sensitive AMP-encapsulin strain in order to demonstrate improved AMP release in the latter system.

Example 5 Design of a Control System

The AMP-encapsulin and protease/lysis systems can be coupled to a specific pathogen, quorum-sensing system for P. aeruginosa. The quorum sensing system from P. aeruginosa was primarily chosen as a proof-of-principle because it is highly specific to P. aeruginosa, has been very well studied [36], and has already been adapted for synthetic biology applications [33, 34, 37]. P. aeruginosa has a unique N-acylhomoserine lactone (AHL) quorum sensing molecule, called 3-oxo-C12-homoserine lactone (3OC12HSL), that is specifically and directly sensed by the native transcription factor LasR. Binding of 3OC12HSL to LasR induces dimerization of LasR, which then is able to bind to and drive expression from pLas1 promoters [36].

The LasR1pLas1 system can be adapted to drive expression of the protease that specifically cleaves the encapsulins and if necessary, a lysis protein to lyse the E. coli Nissle 1917 host and ultimately release AMPS at high concentrations. Encapsulated AMPS can be expressed at high constitutive levels in order to ensure that they will be immediately released at high doses upon lysis. LasR can be expressed at medium constitutive levels to ensure that 3OC12HSL can be detected at any time (FIG. 10, panel A). Established, synthetic sigma70 promoters of different strengths can be used for constitutive expression [38] and these constructs will be inserted into the genome of the E. coli host to prevent variability in expression levels among cells.

In order to minimize premature proteolysis/lysis of the E. coli host, a negative feedback loop logic can be engineered into the protease/lysis cassette (FIG. 10, panel A). Here, the LasR transcription factor will drive expression of an ECF sigma factor (ECFsf) that is orthogonal to the other host E. coli sigma factors [39]. This sigma factor will then activate a unique ECFsf promoter to drive expression of the protease/lysis cassette as well as an anti-sigma factor. The anti-sigma factor will bind and inhibit the ECFsf to prevent further expression of the protease/lysis cassette. Thus, the protease/lysis cassette will only have sustained expression under high concentrations of 3OC12HSL, where the ECFsf is able to out-compete the anti-sigma factor. Alternatively, the anti-sigma factor can be constitutively expressed in order to create a threshold-gated switch, where a set concentration of ECFsf would need to accumulate to overcome the anti-sigma factor. Here, the expression levels of the anti-sigma factor would set the desired threshold. Constitutive expression of the anti-sigma factor also has the added benefit of reducing leaky expression from ECF promoters, which can also lead to premature lysis [39].

Example 6 Optimization and Testing of the System

Initial experiments can be conducted with fluorescent protein reporters (e.g., mVenus) [40] instead of the AMP-encapsulin and protease/lysis cassettes. With these reporters, the concentrations of 3OC12HSL needed to turn on different components of the circuit can be tested and these concentrations can be correlated to cell densities of P. aeruginosa [37]. Further optimization of this system can be performed to obtain the correct degree of expression at an appropriate 3OC12HSL concentration, which may involve altering any of the following: lifetimes of the components via ssrA degradation tags [41], translational production levels via changes in the ribosome binding site (RBS) [42], and copy number of the anti-sigma factor gene to enable sufficient inhibition of the ECFsf. Computational modeling of the rates of production and degradation of the system components will be performed to guide and improve design, according to current synthetic biology practices [43]. Then, the system can be tested using the actual AMP-encapsulin and protease/lysis gene cassettes. The amounts of AMP released into the medium over time can be measured using SDS-PAGE/Western blots as well as the degree of lysis using optical density at 600 nm and microscopy. These measurements can establish whether the system can efficiently deliver a high dose of AMPs. Experiments with P. aeruginosa and exogenously added AMPs can be conducted to determine if the amounts of AMPs released by the system are sufficient to kill the pathogen.

In some cases, the constitutive production of the AMP-encapsulins may be detrimental to the overall fitness of the host E. coli cell, either due to incomplete encapsulation or metabolic burden. In these cases, the system can be designed to only produce the AMP-encapsulin when 3OC12HSL is detected and subsequently lyse the cells after an appropriately spaced delay period at high 3OC12HSL levels (FIG. 10, panel B). The ECFsf downstream of the AMP-encapsulin can be expressed and the stability of the ECFsf (via ssrA tags) can be adjusted to only drive expression of the protease/lysis cassette at high 3OC12HSL levels, which may require further optimization.

Example 7 A Sense-Control-Release System for Clostridium difficile

A sense-control-release system for Clostridium difficile can also be developed based on recent studies identifying a putative, two-component, quorum-sensing system that can sense a unique C. difficile autoinducing peptide (AIP) and activate toxin production in the virulent form of C. difficile [44]. This system can replace the P. aeruginosa LasR sensing component in the sense-control-release system to specifically detect virulent C. difficile instead of P. aeruginosa and release a therapy in response. Because C. difficile is a gram-positive bacterium, the system need to be expressed in a gram-positive host, such as the probiotic gut organism, Lactococcus lactis, which has already been bioengineered for therapy applications [45, 46].

Example 8 Testing of Therapeutic Delivery System Efficacy In Bacterial Liquid Culture

The efficacy of the encapsulated AMP delivery system in killing P. aeruginosa can be tested. Initial studies are performed in bacterial liquid culture of planktonic P. aeruginosa cells (FIG. 12). Different concentrations of the therapeutic E. coli can be mixed with different concentrations of P. aeruginosa and the degree of killing of the P. aeruginosa can be measured by Bactolight live/dead staining (Thermo), OD600, and/or colony forming unit (CFU) counting over time. The E. coli can be differentiated from the P. aeruginosa in microscopy experiments by expressing a fluorescent protein marker in the E. coli. From these experiments, the number of E. coli cells required to kill a certain population of P. aeruginosa can be determined. As controls, two E. coli strains can be constructed that express the following in response to 3OC12HSL detection: 1) secreted AMPs and 2) non-encapsulated AMPs followed by lysis. The ability of these control strains to kill P. aeruginosa can be compared to that of the encapsulated AMP delivery system, in order to demonstrate improved killing in the latter system.

Example 9 Testing of Therapeutic Delivery System Efficacy In Biofilm Model

The ability of the therapeutic E. coli to kill biofilms of P. aeruginosa can also be tested (FIG. 13). Biofilms of P. aeruginosa are commonly found in infections [47]. Biofilms can be grown on pegs of polystyrene attached to a lid of a microtiter plate according to standard practices [48]. Biofilms can then be treated with different concentrations of the therapeutic E. coli by immersing the biofilm pegs in E. coli culture dispensed in the wells of a microtiter plate. The degree of biofilm dispersal can be assayed over time using standard crystal violet staining followed by absorbance measurements at 595 nm [49] as well as live/dead staining and/or CFU counting. Control strains described for the liquid culture can also be tested to demonstrate improved killing in our AMP-encapsulin strain.

Example 10 Engineered Therapeutic Cells for Improved Killing In Biofilm Model

AMPs have been shown to be more effective against bacterial biofilms compared to traditional small molecule antibiotics due their ability to act on non-growing cells as well as inhibit biofilm formation by preventing adhesion and the production of biofilm components (i.e., EPS) [50]. However, there is a possibility that the AMPs released by the E. coli are ineffective against the biofilm because they are not able to penetrate the EPS/DNA matrix to access the P. aeruginosa cells. In this case, a separate strain of E. coli can be engineered that will release DNasel or alginate lyase (AL) via lysis in response to P. aeruginosa detection in order to break up the biofilm. Both DNasel and AL have been shown to be effective in dispersing P. aeruginosa biofilms [51, 52]. The AMP-encapsulin gene cassette can be swapped for the genes for DNasel or AL to create the new strains. The DNaseI/AL strain and the original AMP-encapsulin strain can be mixed together and tested to determine if the combination treatment is more effective in killing P. aeruginosa using the methods previously described.

In order for the therapeutic E. coli to find and stay localized at the biofilm long enough for efficient therapeutic release, the E. coli can be engineered to chemotax toward the 3OC12HSL, using the method developed by Chang and coworkers [34], where LasR was designed to drive expression of cheZ, which promotes smooth swimming toward a metabolite of interest in a ΔcheZ genetic background. Improved killing with the additional cheZ system can be demonstrated using the methods previously described.

Example 11 Testing of Therapeutic Delivery System Efficacy In Host Tissue Culture Model

The ability of our therapeutic E. coli to kill P. aeruginosa can also be tested in a host tissue culture model (FIG. 143). A co-culture of P. aeruginosa with intestinal epithelial cells can be treated with different concentrations of the therapeutic E. coli. In addition to assessing killing of the P. aeruginosa by live/dead staining and/or CFU counting, the fitness of the intestinal epithelial cells can also be tested by standard MTT assays [53] to determine if lysis of the E. coli or P. aeruginosa is detrimental to the host cells. The correlation between number of lysed cells and lower fitness of host cells, if any, can be quantified. If lysis of the E. coli results in significant loss of host cell fitness, several gene deletions in the E. coli strain can be made, which force the cells to only produce lipid IV_(A), instead of lipid A, the component of bacterial LPS that is responsible for endotoxic activity; lipid IV_(A), a precursor to lipid A has been shown to lack endotoxic activity [54]. While these deletions have been demonstrated to effectively reduce the endotoxic effect of E. coli [54], these deletions may reduce the fitness of the E. coli cells, which will need to be assessed to determine they have any effect on the use of our therapeutic E. coli.

Example 12 Growth and Expression of Constructs 124, 125, and 133 in C43(DE3) E. coli Cells

In this example, engineered microcompartment proteins are constructed and expressed in C43 (DE3) E. coli cells.

As shown in FIGS. 14A-C, the gene sequence for Apidaecin Ia peptide (Ap) was fused to the 3′ end of gene sequences for various encapsulin (Encap) constructs. The gene fusions were placed under the control of a T7 promoter in the commercially available pET24a vector. These gene fusions include those that express the following proteins: 1) Ap fused to Encapsulin containing a TEV protease cleavage site and His-tag after position K138 (Ap-Encap(K138) from pMCY124) (FIG. 15A); 2) Ap fused to Encapsulin containing a TEV protease site after position K71 and the TEV protease site and His-tag after position K138 (Ap-Encap(K71,K138) from pMCY125) (FIG. 15B); and 3) Ap fused to Encapsulin containing a TEV protease site after position D60 and the TEV protease site and His-tag after position K138 (Ap-Encap(D60,K138) from pMCY133) (FIG. 15C).

In the constructs herein described, a linker comprised of a Tobacco Etch Virus (TEV) protease site (sequence: ENLYFQG) followed by a GTS (Gly-Thr-Ser) linker is placed between the Ap peptide and encapsulin monomer, in order to enable later cleavage of Ap from encapsulin via TEV protease.

In constructs pMCY125 and pMCY133, within the encapsulin monomer, two specific protease sites are also inserted to enable digestion of the encapsulin cage.

In pMCY125, the first site is inserted following amino acid residue K71 in the encapsulin monomer. This site is chosen because of its location between the surface-accessible E-loop and P-domain of the encapsulin structure. Cleavage at this location was predicted to disrupt the structure of the encapsulin cage. A TEV protease site surrounded by double Gly (Gly-Gly) linkers on both sides of the site is inserted following residue K71. The second site was inserted following amino acid residue K138. This site was shown to be surface-accessible and insertion of a His₆-tag at this location allowed for Ni-NTA affinity purification of the encapsulin cage [55]. A TEV protease site was inserted directly adjacent to the His₆-tag on the N-terminal side. Thus, the following sequence was inserted following residue K138: a pentaglycine linker (Gly_(n)), a TEV protease site, a His₆-tag, and another pentaglycine linker. The His₆-tag was used for affinity purification of the Ap-EncapK71K138 construct. The codon-optimized nucleotide sequence for pMCY125 (SEQ ID NO: 18) is also shown in FIG. 15A.

As controls, gene fusions expressing the following proteins were also prepared: 1) Ap fused to a TEV protease cleavage site alone (Ap-TEV from pMCY126); 2) Ap fused to thioredoxin (Ap-Trx from pMCY117); and 3) Ap fused to the C-terminus of Encapsulin containing the TEV protease site and His-tag after position K138 (Encap(K138)-Ap from pMCY123—Here, Ap peptide would be outward facing in the Encapsin compartment, making it potentially toxic and susceptible to proteolysis.) All gene constructs are diagramed in FIG. 16. The peptide Apidaecin Ia has a sequence of SEQ ID NO: 19: GNNRPVYIPQPRPPHPRIENLYFQ

All DNA plasmids containing the gene constructs were transformed into C43(DE3) E. coli cells (Lucigen) and expression of the corresponding protein constructs were tested. Cells were grown in 10 mL of Luria-Bertani (LB) medium in 50-mL flasks to mid-log phase (optical density at 600 nm (OD600) of 0.4). Isopropyl f3-D-thiogalactoside (IPTG) was then added to the culture to a final concentration of 0.1 mM and culture was grown for another 4 h at 30° C. to induce protein expression. After induction, cells were harvested and resuspended in 400 uL of lysis buffer containing 60% BPER-II detergent (Thermo-Fisher) in buffer A (50 mM Tris pH8.0, 500 mM KC1, 12.5 mM MgCl₂) supplemented with 0.1 mg/mL lysozyme and 10 U/mL of DNaseI. Cells were lysed by incubating at 4° C. with occasional mixing for 15 min. Cells were then centrifuged at 12,000 g, 4° C. for 25 min and the supernatant was collected as the soluble fraction. Samples prior to centrifugation were saved as the “whole” samples, while the supernatant after centrifugation was saved as the “soluble” samples.

All samples were resolved on an any-kDa SDS-PAGE gel (Bio-rad) and stained with Coomassie blue (FIG. 17). Arrows denote predicted location of the expressed protein. Samples expressing fusions with Ap on the N-terminus of Encapsulin are highlighted within the box. Only these fusions show high expression of soluble, induced protein (only soluble protein is available for purification). All control samples do not have detectable expression of the given protein construct.

Example 13 Purification of Constructs 124, 125, and 133

Purification of the protein constructs Ap-Encap(K138), Ap-Encap(K71,K138), and Ap-Encap(D60, K138) described in Example 12 is performed in this example.

Protein was purified using Ni-NTA affinity resin that binds to the His-tag on the Encapsulin constructs. Cells from 50 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) supplemented with 10 mM imidazole and 10 U/mL of DNaseI. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4° C. for 25 min. The supernatant (soluble) was isolated, added to 250 uL of Ni-NTA resin (Qiagen), and equilibrated with the resin at 4° C. for 45 min with rocking. The resin was then packed into a column and the flow through (FT) was collected. The resin was then washed with 5×1 mL fraction of 20 mM imidazole in phosphate buffer. Protein was eluted with 7×200 uL fractions of 250 mM imidazole in phosphate buffer (fractions E1-E7). Fractions E2-E7 were pooled for each sample, concentrated, dialyzed into 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.

For each purification, samples of the soluble fraction before purification, the flow through, and elution fractions E1-E7 were resolved on an any-kDa SDS-PAGE and stained with Coomassie blue (FIG. 18).

Based on the gel, only the Ap-Encap(K71,K138) protein bound to the Ni-NTA resin to high affinity and was thus subsequently purified to high purity (-99% pure). The other constructs did not bind well to the column and thus could not be purified to high purity. These results suggest that the Ap-Encap(K71,K138) protein may have an altered structure compared to the other two proteins, allowing for greater accessibility of the His-tag for purification.

Final yields from the purification are shown in Table 3.

TABLE 3 Yields from the purification for constructs 124, 125 and 133 Theoret. Ap mg/ total total L mg/L mg/L Construct mL uL mg culture culture culture 124 = Ap- 1.02 300 0.307 0.05 6.1 0.51 Encap(K138) 125 = Ap- 5.40 280 1.51 0.05 30.3 2.5 Encap(K71, K138) 133 = Ap- 1.38 260 0.359 0.05 7.2 0.58 Encap(D60, K138)

In Table 3, the columns from left to right show: construct name, concentration of the purified protein in mg/mL, total volume in uL, total amount in mg, total volume of culture that the protein originated from in L, yield of protein in mg of protein per L of original culture, and theoretical yield of the Ap peptide after protease cleavage assuming 100% proteolysis in mg of Ap peptide per L of original culture. The Ap-Encap(K71,K138) protein clearly has the highest yield, nearly 5 times higher than the other proteins.

Example 14 TEV Protease Cleavage of the Purified AP-Encapsulin Fusions

In this example, experiments were conducted to show TEV protease cleavage of the purified Ap-Encapsulin fusions.

60 ug of purified material was digested with 30 U of TEV protease enzyme in a 80 uL reaction with 1 mM DTT and lx TEV protease buffer from Promega. Aliquots of digested material were removed after 1 h, 3 h, and 20 h of digestion at 30 oC. A sample without TEV protease was also prepared as the 0 h sample.

All samples were analyzed by SDS-PAGE using both an any-kDa gel to analyze fragment >15 kDa (FIG. 19A) and a 16.5% Tris-Tricine gel to analyze fragments <15 kDa (FIG. 19B). Samples were also analyzed by Western blot using an anti-TEV site antibody (FIG. 19C). Arrows on the gels above show digested fragments.

P (light gray arrow) denotes TEV protease. U (light gray arrow) denotes undigested material. Underlined numbers (white arrows) denote partially digested material containing Ap peptide. Italicized numbers (dark gray arrows) denote partially digested material NOT containing Ap peptide. Bold numbers (black arrows) denote fully digested material. Table 4 below shows the predicted protein fragments expected from digestion. The numbers are the expected fragment sizes in kDa. The numbers in parentheses correspond to the numbers shown on the gels and Western blot.

TABLE 4 Predicted protein fragments from digestion Degree of digestion Construct 124 125 133 Undigested Ap-Encap1-Encap2- 36.2 37.2 37.3 Encap3 partial Encap1-Encap2- 34.2 (5) 34.3 digestion Encap3 Ap-Encap1-Encap2- 21.4 (6) 21.5 (12) TEV Encap2-Encap3 24.7 26.1 Encap1-Encap2-TEV 33.2 18.4 (7) 18.5 (13) Ap-Encap1-TEV or 20.4 (1) 12.5 11.2 Ap-Encap2-TEV full Encap3 15.8 (3) 15.8 (8) 15.8 (14) digestion Encap2-TEV 17.4 (2) 8.9 (10) 10.3 (15) Encap1-TEV 9.5 (9) 8.2 Ap-TEV 3.0 3.0 (11) 3.0

The data of Table 4 show that construct 125 (Ap-Encap(K71, K138)) is digested by TEV protease to near completion, which is most clearly observed by the disappearance of undigested material (U) and band (6) denoted by the white arrow and the appearance of the Ap peptide band (11) after 20 h digestion. The other constructs do not have clear disappearance of undigested bands denoted by the white arrows based on Western blot, suggesting that they are not well digested by TEV protease. It is also noted that the amount of construct 125 is much higher than the other 2 constructs. Therefore, in the Western blot, the proportion of the band denoted by the white arrow relative to the original is much lower than the other constructs, suggesting nearly complete digestion.

Example 15 Expression of Tandem Ap Peptides Fused to Encapsulin in C43 (DE3) E. coli Cells

This example shows expression of tandem Ap peptides fused to the Encapsulin construct with insertions after K71 and K138.

Gene cassettes expressing up to 4 Ap peptides fused to a single Encapsulin construct were made (FIG. 20A). TEV protease cleavage sites were placed between each Ap peptide as well as prior to the first Ap peptide and prior to the Encapsulin construct in order to ensure the same Ap peptide sequence is obtained upon full protease digestion. The sizes of the tandem Ap peptides that were fused to the Encapsulin construct are as follows: 1xAp=32 amino acids; 2xAp=57 amino acids; 3xAp=82 amino acids; 4xAp=107 amino acids. The gene cassettes were placed under the control of a T7 promoter in the commercially available pET24a. The DNA plasmids were transformed in C43(DE3) E. coli cells and expressed under the same conditions as described above. Cells were lysed and soluble fractions were also obtained in the same manner as described above.

All “whole” and “soluble” samples were resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue (FIG. 20B). Arrows denote predicted location of the expressed protein. Only 1xAp and 2xAp peptide fused to Encapsulin could be expressed. The 3xAp and 4xAp peptide-Encapsulin fusions were not detectable by SDS-PAGE. These data suggest that up to 57-amino acid peptides could be fused to Encapsulin in order to achieve expression. An 82-amino acid peptide fused to Encapsulin could not be expressed, suggesting this fusion and fusions with larger peptide attachments can not properly form Encapsulin microcompartments in order to achieve expression.

Example 16 Expression of Tandem HBCM-2 (HB) Peptides Fused to Encapsulin In C43(DE3) E. coli Cells

This example describes the expression of tandem HBCM-2 (HB) peptides fused to the Encapsulin construct with insertions after K71 and K138. The HBCM-2 peptide has a sequence of SEQ ID NO: 9 from Table 1.

Gene cassettes expressing up to 3 HB peptides fused to a single Encapsulin construct were made as well as a control of HB fused to thioredoxin (Trx) (FIG. 21A). The sizes of the tandem HB peptides that were fused to the Encapsulin construct are as follows: 1xHB=33 amino acids; 2xHB=66 amino acids; 3xHB=99 amino acids. The gene cassettes were placed under the control of a T7 promoter in the commercially available pET24a. The DNA plasmids were transformed in C43(DE3) E. coli cells and expressed under the same conditions as described above. Cells were lysed and soluble fractions were also obtained in the same manner as described above.

All “whole” and “soluble” samples were resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue (FIG. 21B). Arrows denote predicted location of the expressed protein. Only 1xHB and 2xHB peptide fused to Encapsulin could be expressed. The 3xHB peptide-Encapsulin and HB-Trx fusions were not detectable by SDS-PAGE. These data suggest that up to 66-amino acid peptides could be fused to Encapsulin in order to achieve expression. A 99-amino acid peptide fused to Encapsulin could not be expressed, suggesting this fusion and fusions with larger peptide attachments cannot properly form Encapsulin microcompartments in order to achieve expression.

These data confirm the observation at HB can only be expressed when fused to Encapsulin, but not to Trx, demonstrating that only Encapsulin has a toxicity/proteolysis shielding effect that allows HB to be expressed.

Example 17 Design of HB-Enc Constructs with TEV Protease Cleavage Site Insertions

HB-Enc constructs with TEV protease cleavage site insertions were designed based on the following approach.

To express HBCM2 within the lumen of an encapsulin cage, the peptide was directly fused to the N-terminus of the Enc monomer, which is luminal-facing based on the X-ray crystal structure of the encapsulin cage [56]. This strategy was chosen to maximize incorporation of expressed peptide into the Enc cage. Targeting sequences that associate with the lumen of the Enc cage have been identified for loading interior protein [56, 57]. However, quantitative loading using targeting tags in this and other protein compartment systems is often incomplete in heterologous systems and remains a significant challenge [58] [57, 59-61] Direct fusion is expected to ensure each peptide is associated with Enc monomer to maximize loading into the Enc cage.

To isolate HBCM2 following purification of the Enc cage, TEV protease recognition sites were placed between the peptide and the Enc monomer as well as at several surface accessible locations to encourage cage disassembly (FIGS. 22A-B). Kang and coworkers [55] previously demonstrated that a His₆-tag could be placed after exterior residue K138 (EncK138^(His)) and was sufficiently surface exposed for purification via Ni-NTA chromatography. Thus, a construct, HB-EncK138^(TEV-His), containing the following features was initially designed: 1) HBCM2, a TEV site, followed by a G₄T-linker fused to the N-terminus of Enc; and 2) a TEV site followed by a His₆-tag inserted after residue K138 with G₅-linkers flanking both ends of the insert.

Additional sites within the Enc monomer were also identified, which would be surface accessible and amenable to insertion with minimal disruption to the cage structure by examining the conservation of each residue and its flexibility (RMSFA2) based on a reported crystal structure PDB: 3DKT. Residues within the loop of the E-domain of Enc were found to be surface accessible with the highest degree of flexibility and relatively low conservation. Residues D60 and V57 were chosen for insertion because they are at the middle and start of the loop, respectively. Additionally, K71 was also chosen for insertion because its location at the end of the E-domain, immediately preceding the P-domain was thought to hold promise for cage disassembly; the residue is exposed to the exterior and has minimal conservation.

Thus, HB-Enc fusions were constructed with a TEV site flanked with G₂-linkers following residues D60, V57 or K71, in addition to the TEV-His insertion following residue K138. These constructs are referred to as HB-EncD60^(TEV)K138^(TEV-His), HB-EncV57^(TEV)K138^(TEV-His), and HB-EncK71^(TEV)K138^(TEV-His), respectively (FIG. 22A).

Example 18 Expression of HB Peptide Fused to Different Engineered Encapsulin (Enc) Constructs Compared to HB Peptide Fused to Other Common Carrier Proteins In C43(DE3) E. coli

The constructs comprising HB peptide designed as indicated in Example 17 were tested in comparison with constructs where HB peptide is fused to other common carrier proteins. In particular, expression of N-terminal HB-fusions to the following other common carrier proteins with C-terminal His-tags was also tested: Small ubiquitin-like modifier (SUMO), thioredoxin (Trx), glutathione S-transferase (GST), and maltose-binding protein (MBP).

The amino acid sequences of the tested constructs are shown in FIGS. 22B.

In particular, in order to perform the testing, all DNA plasmids were transformed and expressed in C43(DE3) cells as described in Example 12, except that cells were grown in 25 mL of LB medium in 150-mL flasks. IPTG induction was conducted overnight for 17 h at 18° C. to enhance soluble protein expression. Cells were harvested and lysed also as described in Example 12.

All N-terminal HB-Enc constructs showed over-expression of soluble HB-Enc protein by SDS-PAGE and anti-His-tag Western blot (FIG. 23A). In contrast, a C-terminal Enc-HB fusion was not expressed as soluble protein. The HB-EncK71^(TEV)K138^(TEV-His) construct showed the most robust expression with the highest cell density (OD₆₀₀ of 5.8) (FIG. 23B), compared to the other double TEV site containing constructs (OD₆₀₀ of 3.7-4.0). Removal of the K138^(TEV-His) site from this construct to produce HB-EncK71^(TEV) maintained this higher level of expression and cell density (OD₆₀₀ of 5.6), which was also similar to the EncK138^(His) control.

Expression of N-terminal HB-fusions to the following other common carrier proteins with C-terminal His-tags was also tested: Small ubiquitin-like modifier (SUMO), thioredoxin (Trx), glutathione S-transferase (GST), and maltose-binding protein (MBP) (FIG. 23B). These constructs had minimal expression in C43(DE3) cells. Only HB-SUMO could be detected by SDS-PAGE, while all other constructs were only detected by anti-His-tag Western blot.

Expression of HB-Trx, HB-GST, and HB-MBP were found to produce truncated products, demonstrating that they are sensitive to proteolysis in cell lysate. Given that the truncated products can be detected by anti-His-tag and are nearly full length (<5 kDa difference) indicates that proteolysis occurred on the N-terminus of the protein close to or within the HBCM2 sequence. Expression of EIB-EncK71^(TEV)K138^(TEV-His) also showed some truncated products by Western blot, but degradation was minimal compared to HB-Trx, HB-GST, and HB-MBP. All other HB-Enc fusions did not show truncated products. HB-EncK71^(TEV) cannot be detected by anti-His-tag given its lack of a His-tag.

The above results indicate that HB-Enc fusions exhibit robust expression in E. coli C43(DE3) cells compared to other HB-carrier protein fusions

In particular, the expression data herein described reveals that HB peptide requires fusion to Enc for robust expression in C43(DE3) cells

Example 19 Purification and TEV Protease Digestion of HB-Enc Fusions

Constructs HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), HB-EncV57^(TEV)K138^(TEV-His) were purified as follows. Cells from 50 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) and 10 U/mL of DNasel. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4° C. for 25 min. The supernatant (soluble) was isolated and then heated at 85° C. for 15 min. The material was then centrifuged at 12,000 g, 4° C. for 10 min and the soluble fraction was collected. Constructs were further purified by addition of ammonium sulfate to 25%(w/v) followed by centrifugation at 12,000 g, 4° C. for 25 min to collect the insoluble pellet. Purified material was re-suspended in 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.

Construct HB-EncK71^(TEV) was purified as follows. Cells from 50 mL of culture were harvested and resuspended in 60% B-PER II in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl), supplemented with 0.1 mg/mL lysozyme and 10 U/mL DNase. Cell lysate was incubated at 4° C. for 1 h to lyse the cells. The supernatant (soluble) was isolated and then heated at 70° C. for 15 min. The material was then centrifuged at 12,000 g, 4° C. for 10 min and the soluble fraction was collected. The soluble fraction was dialyzed overnight at 4° C. into phosphate buffer to remove the B-PER. The material was further purified by addition of ammonium sulfate to 50%(w/v) followed by centrifugation at 12,000 g, 4° C. for 25 min to collect the insoluble pellet. Purified material was re-suspended in 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.

Construct HB-EncK71^(TEV)K138^(TEV-His) was partially purified Ni-NTA chromatography. Cells from 50 mL of culture were harvested and resuspended in 60% B-PER II in phosphate buffer (25 mM sodium phosphate pH 7.5, 100 mM NaCl), supplemented with 0.1 mg/mL lysozyme and 10 U/mL DNase. Cell lysate was incubated at 4° C. for 1 h to lyse the cells. The supernatant (soluble) was isolated, added to 250 uL of Ni-NTA resin (Qiagen), and equilibrated with the resin at 4° C. for 45 min with rocking. The resin was then packed into a column and the flow through (FT) was collected. The resin was then washed with 5×1 mL fraction of 5 mM imidazole in phosphate buffer. Protein was eluted with 7×200 uL fractions of 250 mM imidazole in phosphate buffer (fractions E1-E7). Fractions E2-E7 were pooled for each sample, concentrated, dialyzed into 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.

B-PER II was found to at least partially prevent proteolysis of HB-EncK71^(TEV) and HB-EncK71^(TEV)K138^(TEV-His), whereas many different protease inhibitors including pepstatin, EDTA, PMSF and Roche protease inhibitor cocktail were not effective.

All purified constructs were digested by addition of exogenous TEV protease, followed by overnight incubation at 4° C. Products were resolved on a 16.5% Tris-Tricine gel (Bio-Rad). All constructs were found to at least partially release HBCM2 peptide (FIG. 24). HB-EncK71^(TEV)K138^(TEV-His) and HB-EncK71^(TEV) released the most peptide with apparent complete digestion based on the sizes of the fragments observed after digestion. In contrast based on densitometry analysis, HB-EncK138^(TEV-His), EncD60^(TEV)K138^(TEV-His), and HB-EncV57^(TEV)K138^(TEV-His) released fewer peptide per fusion protein. These constructs had a relative HB/HB-Enc ratio of 0.26 to 0.56, assuming that the HB/HB-EncK71^(TEV)K138^(TEV-His) is 1.

HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His,) HB-EncV57^(TEV)K138^(TEN-His) constructs with a GT linker between the TEV site and the N-terminus of Enc, instead of the flexible G₄T linker, were also purified and tested for digestion for TEV protease digestion. However, the HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), and HB-EncV57^(TEV)K138^(TEV-His) with the shorter GT linker did not result in detectable release of HB peptide by SDS-PAGE (FIG. 25). These results suggest that the G₄T linker is crucial to release of peptide for these constructs.

Example 20 Insertion of TEV Protease Site at Position K71 Disrupts Cage Formation Which Enables Highly Efficient Release of Peptide by TEV Protease

K71^(TEV) containing constructs as well as its susceptibility to proteolysis, all of the HB-Enc constructs were analyzed by size-exclusion chromatography (SEC) and native PAGE to test for cage formation (FIGS. 33A and 33B). SEC analysis revealed that HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), HB-EncV57^(TEV)K138^(TEV-His), and the control EncK138^(His) constructs primarily contain a high molecular weight (MW) species that migrated close to void volume, indicative of cages. In contrast, the majority of HB-EncK71^(TEV)K138^(TEV-His) migrated as lower MW species. HB-EncK71^(TEV) appeared to contain some high MW species, but primarily consisted of the lower MW species. These results were confirmed by Native PAGE which can also be used to test for cage formation. Protein is resolved by native PAGE on an any-kDa gel (Bio-Rad) in the absence of SDS in the running and loading buffers.

All purified HB-Enc constructs (with G₄T linker) were analyzed by native PAGE (FIG. 26). Native PAGE analysis revealed that HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), HB-EncV57^(TEV)K138^(TEV-His), and the control EncK138^(His) constructs primarily contain a high molecular weight (MW) species that did not enter the gel, which is indicative of cages. In contrast, the majority of HB-EncK71^(TEV)K138^(TEV-His) migrated as lower MW species that entered the gel. HB-EncK71^(TEV) appeared to contain some high MW species, but primarily consisted of the lower MW species.

The high and low MW species can be isolated by size exclusion chromatography (SEC) on a SHIMADZU FPLC with a Superose 6 increase 3.2/300 column. Typically, 50 μL of a 1 mg/mL protein solution was loaded onto the column. Protein was eluted with 50 mM NaH₂PO₄, pH 8.0, 200 mM NaCl at a flow rate was 0.2 mL/min for 20 min. Species migrating at a retention time of 7-8 min were collected as the high MW species. Species migrating at a retention time of 10-11 min were collected as the low MW species. Example SEC traces for Ap-EncK138^(TEV-His) and Ap-EncK71^(TEV)K138^(TEV-His) can be found in FIG. 27.

Species can be analyzed by transmission electron microscopy (TEM). Purified material, 10 μL at a concentration of ˜0.1-0.25 mg/mL, was spotted on a Cu grid coated with carbon type B-formvar for 3 min. Material was wicked away using filter paper. Grid was washed once with 10 μL of water for 1 min, then wicked away. Grid was then stained with 2% uranyl acetate in water for 3 min. Stain was wicked away and grid was washed an additional two more times with 10 μL of water for 1 min each wash. Grids were dried at room temperature for at least 1 h prior to TEM. Grids were imaged on a FEI Titan TEM at 80 kV.

Isolation of the high and low MW HB-Enc constructs after SEC followed by transmission electron microscopy (TEM) analysis revealed that the high MW species indeed were comprised of encapsulin cages, while no cages were observed for the low MW species (FIG. 28). All observed cages were ˜25 nm in diameter, with no significant difference observed among the different HB-Enc constructs.

Similar results for Native PAGE and TEM analyses were obtained for Ap-Enc fusions (FIG. 29).

Example 21 Cage Forming HB-Enc Constructs are Resistant to Proteolysis In Cell Lysate

To test for proteolysis in cell lysate, cells expressing the HB-Enc constructs from 25 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) and 10 U/mL of DNaseI. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4° C. for 25 min. The supernatant (soluble) fraction was collected and incubated at 4° C. overnight. Samples for SDS-PAGE analysis were collected immediately after lysis and after overnight incubation.

As controls, samples after BPER-II lysis were also prepared. Samples were resolved on an any-kDa SDS-PAGE gel (Bio-Rad) and stained with Coomassie blue. For Western blot analysis, samples were resolved on an any-kDa and blotted to a PVDF membrane using a Transblot Turbo System (Bio-Rad) at 2.5 A for 7 min. Samples were probed using a mouse anti-His-tag primary antibody and a rabbit anti-Mouse-HRP conjugated secondary antibody (both from Bio-Rad). HRP was detected on the membrane using chemiluminescence.

All three cage-forming constructs (HB-EncK138^(TEV-His), HB-EncD60^(TEV)K138^(TEV-His), and HB-EncV57^(TEV)K138^(TEV-His)) were resistant to proteolysis (FIG. 30). Full length product was observed even after overnight incubation with minimal degradation products. In contrast, non-cage forming HB-EncK71^(TEV)K138^(TEV-His) and HB-EncK71^(TEV) were both significantly degraded immediately after French press lysis and completely proteolyzed after overnight incubation. Given the detection of degradation products by anti-His-tag, proteolysis appears to primarily occur at the N-terminus of the HB-Enc fusion, degrading the HB peptide.

These results show that cage-forming HB-Enc constructs are capable of protecting the HB peptide from proteolysis, which has also been observed in other protein compartment systems [62]. In contrast, the non-cage forming, K71^(TEV) containing HB-Enc constructs are susceptible to proteolysis, which very likely accounts for their ability to be completely digested by TEV protease. Interestingly, these constructs are able to be highly over-expressed in C43(DE3) E. coli compared to other HB-carrier protein fusions, suggesting that the K71^(TEV) containing Enc constructs still enable HB expression, perhaps by partial occlusion of the peptide to prevent it from carrying out its toxic mode of action.

Example 22 Released HB-TEV Peptide has Anti-Bacterial Activity Against E. Coli, While Ap-TEV Peptide is Inactive

The anti-bacterial activity of HB-EncK71^(TEV)K138^(TEV-His) and TEV protease-digested HB-EncK71^(TEV)K138^(TEV-His) were tested for growth inhibition activity against BL21(DE3) E. coli cells

Growth inhibition activity can be tested against E. coli BL21(DE3) cells. Overnight cultures of cells were diluted to 200 μL of an initial OD₆₀₀ of 0.05-0.1 in LB medium. Diluted cultures were grown at 37° C., 1000 rpm in a PHMP-4 Microplate Shaker (Grant Instruments, Cambridge, England). Protein was added to the culture at an OD₆₀₀ of 0.1-0.2. Growth measured by OD₆₀₀ was monitored over time and were conducted in triplicate. TEV-digested material contained HB-Enc fusion protein (0.5 mg/mL) that was incubated with TEV protease (0.5 U/mL) overnight for 17 h at 4° C.

The results are shown in FIG. 31, panel A. The HB-EncK71^(TEV)K138^(TEV-His) fusion protein did not inhibit the growth of E. coli at a concentration of 40 μg/mL. However, TEV protease-digested EncK71^(TEV)K138^(TEV-His) showed significant bacteriostatic activity (theoretical HB peptide released was 4 μg/mL). A control with the addition of TEV protease alone also showed no growth inhibition, confirming that the inhibitory activity was due to released HB peptide.

Growth inhibition assays of chemically synthesized M-HB-TEV peptide (M-HBCM2-TEV, the peptide product that is released by digestion) showed significant anti-bacterial activity at 2 μg/mL (FIG. 31, panel B), consistent with the reported MIC [63], suggesting that the N-terminal Met residue and the residual C-terminal TEV site did not significantly affect activity.

Growth inhibition assays of chemically synthesized native Ap peptide and M-Ap-TEV peptide only showed significant anti-bacterial activity for the native Ap peptide starting at 100 μg/mL. The M-Ap-TEV peptide as inactive against BL21(DE3) E. coli. Thus, the Ap-Enc system serves as a model/case study for a non-active peptide.

Example 23 Expression of HB-Enc Constructs in TOP10 E. coli Cells from an Arabinose Inducible System

All HB-Enc constructs as well as HB-Trx, HB-SUMO, HB-GST, and HB-MBP were cloned under a pBAD promoter for expression in TOP10 E. coli. Expression in TOP10 cells under 40 μM (low) or 10 mM (high) arabinose induction was tested. Overnight cultures of cells were diluted to 200 μL of an initial OD₆₀₀ of 0.1 in LB medium. Diluted cultures were grown at 37° C., 1000 rpm in a PHMP-4 Microplate Shaker (Grant Instruments, Cambridge, England). Arabinose inducer was added to the indicated concentrations once cultures reached an OD₆₀₀ of 0.2. After 4-5 h induction, cells were harvested and lysed as described in [00254] and tested for expression by SDS-PAGE analysis and anti-His₆ Western blot.

HB-Trx, HB-SUMO, and HB-GST had little to no detectable expression by SDS-PAGE analysis in TOP10 E. coli cells under an arabinose-inducible pBAD promoter (FIG. 33A). A partial HB-MBP was expressed under high arabinose induction (10 mM) but was truncated without the HB peptide at the N-terminus (FIG. 33B). In contrast, HB-EncD60^(TEV)K138^(TEV-His), HB-EncK71^(TEV)K138^(TEV-His), and HB-EncK71^(TEV) could all be well expressed in the system under low arabinose induction (40 μM) conditions. Some truncation of the K71^(TEV) containing constructs was observed in the expression gel, but it was not as significant as truncation of HB-MBP.

Despite low expression of HB-Trx, HB-SUMO, and HB-GST and truncated expression of HB-MBP under 10 mM arabinose induction, cells expressing these constructs reached a significantly lower OD₆₀₀ after induction (1.9-2.4) compared to cells significantly over-expressing HB-EncK71^(TEV)K138^(TEV-His) and HB-EncK71^(TEV) constructs at 40 μM arabinose (OD₆₀₀ 2.7) (FIG. 33C). These results suggest that the HB-Trx/SUMO/GST/MBP constructs may be conferring some toxicity to the expression cells, whereas the K71^(TEV) containing HB-Enc constructs confer limited toxicity even when highly over-expressed. Expression of HB-EncD60^(TEV)K138^(TEV-His) consistently results in lower cell density (OD₆₀₀ 2.3) compared to expression of the K71^(TEV) containing HB-Enc fusions, likely associated with some insoluble cage expression.

Overall, these results are similar to the C43(DE3)/T7 induction system, where fusion of HB to the encapsulin constructs enabled its expression and prevented its proteolysis during expression.

Example 24 Expression of Protease-Sensitive HB-Trx, HB-SUMO, HB-GST, and HB-MBP Constructs in BL21(DE3) E. coli Cells from a T7 IPTG Inducible System

HB-Trx, HB-SUMO, HB-GST, and HB-MBP were expressed in BL21(DE3) from a T7 IPTG inducible promoter using the same method as for C43(DE3) cells described in Example 12. Protease sensitivity of the constructs in lysate were tested as described in Example 21.

HB-Trx, HB-SUMO, HB-GST, and HB-MBP were found to be significantly over-expressed in BL21(DE3) E. coli (FIG. 34A).

Despite significant over-expression of these constructs, they are all highly susceptible to proteolysis after cell lysis (FIG. 34B). Immediately following lysis in the presence of B-PER-II, significant proteolysis of HB-GST and HB-MBP were observed by SDS-PAGE, contrasting with the HB-Enc constructs which did not exhibit significant proteolysis in the presence of B-PER (FIG. 30). Following French press lysis in the absence of B-PER, all other HB carrier protein fusions were subjected to rapid proteolysis with complete disappearance of full-length protein after overnight incubation at 4° C.

It is possible that expression of non-Enc HB carrier protein fusions in BL21(DE3) cells is due to rapid protein synthesis relative to the rate of proteolysis in this strain, in contrast to C43(DE3) or TOP10 cells, where protein synthesis is possibly slower than proteolysis.

The data in all three expression systems (C43(DE3)/T7, BL21(DE3)/T7, TOP10/pBAD) demonstrate that fusions of HB to other common carrier proteins are highly susceptible to proteolysis. The non-cage forming HB-Enc fusions are also susceptible to proteolysis, but their expression is robust in all three expression systems, suggesting that 1) they are not as susceptible to proteolysis as the other fusions; and 2) the K71^(TEV) containing Enc proteins may be providing some additional occlusion of the HB peptide to allow its expression. The cage-forming HB-Enc fusions fully protect the HB peptide from proteolysis, but their over-expression is not as robust because they appear to confer some toxicity to the expression strain upon over-expression.

Thus, there appears to be a trade-off between high over-expression and peptide release (non-cage forming HB-Enc) versus protection from proteolysis (cage forming HB-Enc).

Example 25 Enc Constructs Comprising M-Ap-

Ap was fused to the various engineered Enc examined in this study as well as typical carrier proteins SUMO, Trx, GST, and MBP and expression of the fusions from a T7 promoter in E. coli C43(DE3) cells was tested (FIG. 36). Fusions of Ap to the N-terminus of the various engineered Enc proteins were over-expressed in C43(DE3) cells, as well as Ap-SUMO, Ap-GST, and Ap-MBP. However, Ap-Trx and a C-terminal EncK138^(TEV-His)-Ap fusion were not well expressed and could not be detected by SDS-PAGE. These data suggest that Enc works comparably to SUMO, GST, and MBP as a carrier protein for non-toxic Ap, but it must be fused to the N-terminus of Enc to enable Ap expression.

Interestingly, Ni-NTA purification of Ap-EncK71^(TEV)K138^(TEV-His) was achieved with high affinity binding of the protein to the resin (FIG. 37). In contrast, Ap-EncK138^(TEV-His), Ap-EncD60^(TEV)K138^(TEV-His) and Ap-EncV57^(TEV)K138^(TEV-His) did not bind well to the Ni-NTA resin and thus, needed to be purified using an alternative method of heat precipitation followed by ammonium sulfate precipitation. Characterization of the purified fusions by SEC, native PAGE, and TEM showed similar results to the HB-Enc fusions, where K71^(TEV) containing Ap-Enc constructs did not form protein cages, whereas Ap-Enc without the K71^(TEV) insertion were able to form cages (FIG. S8).

Example 26 Isolation of M-Ap-TEV Peptide Following TEV Protease Cleavage

In addition to HBCM2 peptide, engineered Enc constructs were provided including the proline-rich, unstructured AMP, apidaecin Ia (Ap) [64].

The Ap peptide was initially tested as a model antimicrobial peptide with intercellular toxic activity; proline-rich AMPS are generally bacteriostatic by inhibition of the ribosome [65]. However, we later discovered that Ap with a residual C-terminal cleaved TEV site (Ap-TEV) was found to lack bacteriostatic activity, unlike native Ap, suggesting that the residual TEV site interferes with Ap activity. We proceeded to test whether fusion of Enc to Ap helps its expression as a case study for a non-toxic peptide.

In particular the engineered Enc construct Ap-EncK71^(TEV)K138^(TEV-His) wherein 1) a TEV protease site followed by a GGT linker was placed between the C-terminus of Ap and the N-terminus of Enc; 2) a TEV protease site is inserted following residue K71 in Enc with GG-linkers on both the N- and C-termini of the insertion; and 3) a TEV protease site followed by a hexa-histidine tag is inserted following residue K138 in Enc with G₅-linkers on both the N- and C-termini of the insertion.

The engineered Enc construct Ap-EncK71^(TEV)K138^(TEV-His) was tested for expression in C43(DE3) cells.

In particular, purified Ap-EncK71^(TEV)K138^(TEV-His) was digested with TEV protease as described in Example 14. Following overnight digestion at 4° C., digested material was filtered through a centrifugal filter with 10 kDa molecular weight cutoff (Vivaspin, Satorius). Peptide was recovered in the filtrate. Material in the filter (<500 μL) was diluted an additional two times to 5 mL using phosphate buffer (25 mM sodium phosphate pH 7.5, 100 mM NaCl) and centrifuged to collect additional filtrate. All filtrate was pooled and lyophilized. Final purified material was analyzed by SDS-PAGE and quantified by absorbance at 280 nm.

Ultimately, an overall yield of 3.5 mg/L culture of Ap-TEV peptide was obtained from 43 mg/L of Ap-EncK71^(TEV)K138^(TEV-His) fusion protein (FIG. 35). These data collectively show that Enc can aid expression and purification of a non-toxic peptide.

Example 27 Prophetic Example of Engineered Constructs Designed to Allow Post Isolation Digestion of the Residual Protease Cleavage Site Attached to the C-Terminus of the Target Proteins

In an example target protein (abbreviated TP) where a residual protease cleavage site on the C-terminus of TP interferes with the activity of TP, the following methods may be conducted to remove the majority of the residual site, leaving only a proline residue at the C-terminus of TP. Removal of residual site may possibly restore the activity/function of the TP.

First, a fused TP-Enc construct will need to be re-designed such that a proline residue is inserted between the C-terminus of TP and the N-terminus of the adjacent protease cleavage site. The new TP-Enc construct can then be over-expressed in C43(DE3) E. coli cells as described in Example 12, purified as described in Example 19, and digested with an appropriate protease as described in Example 14 and 19.

Following digestion, the released TP can be isolated by size exclusion chromatography or a centrifugal filter with an appropriate molecular weight cutoff. Alternatively, released TP can is isolated using ion exchange chromatography or reverse-phase (e.g., C18) chromatography, if appropriate.

Purified released TP can then be digested with commercially available carboxypeptidase A and/or B, according to manufacturer's instructions. Following carboxypeptidase digestion, TP with a residual proline at its C-terminus can be re-isolated using the same methods as in [00379].

Example 28 Isolation and Detection of Microcompartments from Bacteria

Cages from bacteria can be isolated by re-suspending cells in buffer and lysing the suspended cells. For example, cells can be re-suspended in a buffer such as 50 mM NaH₂PO₄, pH 8.0 with 200 mM NaCl and lysed by French pressure cell at 14,000 psi. Following removal of insoluble material by centrifugation at 12,000 g, 4° C. for 15 min, the supernatant is heated at 70-85° C. for 10 min. Only encapsulin cages will remain soluble under these conditions and insoluble material can again be removed by centrifugation. Ammonium sulfate precipitation at 25 or 50% can be performed to further purify the material, followed by size exclusion chromatography (SEC). SEC can be done using SHIMADZU FPLC with a Superose 6 increase 3.2/300 column (GE biosciences). One to 5 mg of sample is loaded, and protein is eluted with 50 mM NaH₂PO₄, pH 8.0, 200 mM NaCl at a flow rate of 0.2 mL/min for 20 min. Protein cages should elute as a high molecular weight species near the void volume, between 7 and 8 min.

Protein cages collected after SEC can be detected using transmission electron microscopy. For example, protein can be spotted on a copper TEM grid coated with carbon type B-formvar and stained using 2% uranyl nitrate, using standard methods. Grids can be examined on a transmission electron microscope (e.g., FEI Titan) at 80 kV. Hexagonal species of ˜25 nm diameter is indicative of cage formation.

Observation of (1) a high molecular species by SEC and (2) hexagonal cage features by TEM confirms cage formation.

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods may include a large number of optional composition and processing elements and steps.

In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

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1. An engineered microcompartment protein, comprising an encapsulin protein having sequence SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, and comprising a P-Domain (Peripheral Domain), an E-Loop (Elongated Loop) and an A-Domain (Axial Domain), wherein the P-Domain comprises a first fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 26 residues alpha helix α1, linked to a 0 to 22 residues first non-structured region, linked to a 4 to 11 residues alpha helix α2, linked to a 3 to 9 residues beta strand β1, linked to a 3 to 13 residues second non-structured region, a second fragment of the P-Domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising a direction N-terminus to C-terminus a 9 to 15 residues beta strand β4, linked to a 6 to 15 residues alpha helix α3, linked to a 0 to 10 residues first non-structured region, a 18 to 29 residues alpha helix α4, and a 9 to 21 residues second non-structured region, and a third fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded microcompartment protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 10 residues beta strand β9, linked to a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand β10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand β11; the E-Loop has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 8 to 16 residues beta strand β2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand β3, linked to a 0 to 6 residues second non-structured region; and the A-Domain of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 0 to 8 residues beta strand β5, linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix α5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand β36, linked to a 9 to 16 residues alpha helix α6, linked to a 1 to 24 third non-structured region, linked to a 0 to 16 residues alpha helix α7, linked to a 0 to 8 residues fourth non-structured region, linked to a 1 to 10 residues beta strand β7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand β8, linked to a 2 to 12 residues sixth non-structured region; and wherein the P-domain, A-domain and E-loop are arranged together in a configuration comprising, in a direction N-terminus to C-terminus, the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain; the engineered microcompartment protein further comprising, a target protein having an N-terminus, a C-terminus inserted at the N-terminus of the first segment of the P-domain of the encapsulin protein alone or in combination with a tag and/or a linker; at least one first protease cleavage site inserted between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein, alone or in combination with a tag and/or a linker; and at least one second protease cleavage site inserted at an insertion site at the C terminus of the E-loop of the encapsulin protein, within 1 to 17 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein, alone or in combination with a tag and/or a linker to enable digestion of the encapsulin and release of the target protein.
 2. The engineered microcompartment protein of claim 1, wherein the target protein comprises at least one non-native antimicrobial peptide.
 3. The engineered microcompartment protein of claim 2, wherein the at least one non-native antimicrobial peptide is selected from Apidaecin Ia, HBCM-2, cecropins, magainins, melittin, protegrins, and nisins.
 4. The engineered microcompartment protein of claim 1, wherein the target protein has a sequence up to 80 amino acids in length.
 5. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted within the first non-structured region of the E-loop to provide a cage-forming engineered microcompartment protein.
 6. The engineered microcompartment of claim 5, wherein the at least one second protease cleavage site is inserted at positions X57 and/or X66 of SEQ ID NO:
 1. 7. The engineered microcompartment of claim 6, wherein the E-loop has sequence YAAHPLGEVEVLSDENEVVKWGLRK SLP (SEQ ID NO: 59), YTVVPEGRLKKIEDNPGNVCTGMYQVKP (SEQ ID NO: 60), YAAVNTGELRPIDDTPEDVDMKLRQVQP (SEQ ID NO: 61), YAAVNT GRRT ALE DKAEGA S IF QRQVLP (SEQ ID NO: 62), or FSALGTGHVSRVAADTPGVEALQRHVVR (SEQ ID NO: 63).
 8. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted in the first non-structured region of the E-loop, the (35 beta strand of the A-domain and/or the alpha helix cc5 of the A-Domain to provide a cage forming engineered microcompartment protein.
 9. The engineered microcompartment of claim 8, wherein the at least one second protease cleavage site is inserted at any one of positions X132 and X144 of SEQ ID NO:
 1. 10. The engineered microcompartment of claim 8, wherein the A-Domain has sequence LLSF EERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGH YPLEKRVEECLRGGKIITTPRIEDALVVSER (SEQ ID NO: 64), LREGTSNPKLALPSSASDYPAAIAAALNQLRLAGVNGPYAVVLGAGVYTALSGG DDEGYPVFRHIESLIDGKIVWAPAIEGGFVLSTR (SEQ ID NO: 65) LLTEDGIVKFPISNWSEGENPFKDISIGLAKFIENGIVGRKALVVSPNLFVQLQRIQ PGTGTTEYDRINKLLDGNIFSTPVLKDDKAVLVCSE (SEQ ID NO: 66), ILNAEGAQKLQISDWGQGENPYTDIVKAINMIREKGIVGRFVLCLSQSLYFDLQRI QQGTGMTEAQRISSMIGNLYNVPVIKGKKAALICAE (SEQ ID NO: 67), or LLTVKGSSKIKKSDWSQGENSFADITAGVAQLAKTGYLGRYALVVSPDLFLDLQ RLQPNTGLLEIDRIKKLIGDNVYMTSVMGPGKAVLVCAE (SEQ ID NO: 68).
 11. The engineered microcompartment of claim 5, wherein the target protein is a protease sensitive target protein.
 12. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted within the beta-strand β3 to provide a non-cage-forming engineered microcompartment protein.
 13. The engineered microcompartment of claim 12, wherein the at least one second protease cleavage site is inserted at position X71 of SEQ ID NO: 1
 14. The engineered microcompartment of claim 1, wherein the target protein is a protease sensitive target protein and the engineered microcompartment protein further comprises a proline between the N-terminus of the first at least one protease cleavage site and the C terminus of the target protein
 15. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted at any one of positions X57, X60, X71 and X139.
 16. The engineered microcompartment protein of claim 1 wherein the at least one encapsulin protein has a sequence of SEQ ID NO:
 47. 17. The engineered microcompartment protein of claim 1, wherein the at least one encapsulin protein is from the PF04454 protein family.
 18. The engineered microcompartment protein of claim 1, wherein the at least one first protease cleavage site and/or the at least one second protease cleavage site are selected from ENLYFQ\S(orG)) (TEV protease recognition), LEVLFQGP (HRV 3C protease recognition), LVPRGS (thrombin recognition), DDDDK (enterokinase recognition), and IEGR (Factor Xa recognition).
 19. The engineered microcompartment protein of claim 1, wherein the target protein is fused to the encapsulin protein in an insertion region further comprising a linker and/or a tag.
 20. The engineered microcompartment protein of claim 1, wherein the at least one first protease cleavage site is fused to the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein in an insertion region further comprising a linker and/or a tag.
 21. The engineered microcompartment protein of claim 1, wherein the at least one second protease cleavage site is fused to the C-terminus of the E-loop and/or to the N-terminus of the A-domain of the encapsulin protein in an insertion region further comprising a linker and/or a tag.
 22. The engineered microcompartment protein of claim 1, wherein the tag is selected from His-Tag, Strep-Tag, FLAG-Tag, Avi-Tag, E-Tag, HA-Tag, Myc-Tag, and TC-Tag.
 23. An engineered microcompartment comprising at least one engineered microcompartment proteins of claim
 1. 24. A method to produce in a bacterial cell a protein non-native to the bacterial cell, the method comprising introducing into the bacterial cell at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1 in which the target protein is the protein non-native to the bacterial cell; wherein the at least one first polynucleotide is operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the bacterial cell; and wherein the introducing is performed to obtain expression in the bacterial cell of the at least one engineered microcompartment protein to obtain the protein non-native to the bacterial within at least one engineered microcompartment formed by the at least one engineered microcompartment protein.
 25. The method of claim 24, wherein the engineered microcompartment protein is the cage forming engineered microcompartment protein of claim
 5. 26. The method of claim 24, wherein the engineered microcompartment protein is the non-cage forming engineered microcompartment protein of claim
 12. 27. The method of claim 24, wherein the protein non-native to the bacterial cell is a toxic non-native protein capable of causing a cell damage.
 28. The method of claim 24, wherein the protein non-native to the bacterial cell is a non-native protein capable of being degraded within the bacterial cell.
 29. A system to produce, in bacterial cell, a protein non-native to the bacterial cell, the system comprising at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1, wherein the target protein is the protein non-native to the bacterial cell, the at least one engineered microcompartment protein operatively linked to one or more first regulatory elements configured to enable the expression of the at least one engineered microcompartment protein in one or more bacterial cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different engineered microcompartment proteins to form at least one microcompartment within the one or more bacterial cell, the system further comprising at least one of: the one or more bacterial cells capable of expressing the at least one first polynucleotide to provide an expressed engineered microcompartment protein herein described; at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the protease cleavage sites of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and at least one protease capable of targeting the protease cleavage site of the engineered microcompartment protein to release the protein non-native to the bacterial cell from the engineered microcompartment protein in the bacterial cell.
 30. A vector comprising at least one polynucleotide encoding for an engineered microcompartment protein of claim 1, alone or in combination with regulatory elements in accordance with the disclosure.
 31. A bacterial cell comprising at least one engineered microcompartment of claim
 23. 